Compositions and methods for treating disease using Salmonella T3SS effector protein (SipA)

ABSTRACT

The invention provides compositions and methods for reducing one or more symptoms of disease by administering compositions comprising SipA. The invention&#39;s compositions and methods are particularly advantageous in reducing symptoms of diseases that are associated with overexpression of P-gp and/or p53. The invention&#39;s compositions and methods are useful in reducing cancer symptom and/or cancer multidrug resistance (MDR). The invention provides a method for reducing one or more symptoms of cancer in a mammalian subject in need thereof, comprising administering to said subject a composition comprising purified SipA. In one embodiment, said SipA is operably conjugated to a nanoparticle. In another embodiment, said cancer comprises cancer cells resistant to at least one cytotoxin.

This application is a divisional of, and claims priority to, co-pendingU.S. non-provisional application Ser. No. 15/103,844, filed Jun. 30,2016, which is the U.S. national stage filing under 35 U.S.C § 371 of,and claims priority to, international application No. PCT/US14/69707,filed Dec. 11, 2014, which claims priority under 35 U.S.C. § 119(e) toU.S. Provisional Application Ser. No. 61/914,600, filed on Dec. 11,2013, each of which is herein incorporated by reference in its entirety.

GOVERNMENT INTEREST

This invention was made with government support under Grant numberDK056754 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The invention provides compositions and methods for reducing one or moresymptoms of disease by administering compositions comprising SipA. Theinvention's compositions and methods are particularly advantageous inreducing symptoms of diseases that are associated with overexpression ofP-gp and/or p53. The invention's compositions and methods are useful inreducing cancer symptoms and/or cancer multidrug resistance (MDR).

BACKGROUND OF THE INVENTION

Current therapies used to treat disease (e.g., cancer, infection withmicroorganisms, etc.) have considerable limitations. For example,current chemotherapeutics used to treat many cancer patients suffer fromhigh toxicity, poor tumor targeting, and multidrug resistance (MDR),which together often result in incomplete destruction of the tumors.These drawbacks prevent effective treatment and are associated withincreased morbidity and mortality.

The ability of cancer cells to develop resistance to multiplestructurally and functionally non-related cytotoxic drugs, such asmulti-drug resistance, is a major barrier to effective chemotherapy andis a critical unmet need. Over the past two decades, numerousresearchers across many disciplines have investigated multidrugresistance with the ultimate goal of developing novel P-gp modulators asa way to revert MDR in human cancers. Excitement in this field of drugdevelopment is bolstered by several reports documenting many agents,which modulate the function of P-gp are able to restore the cytotoxicityof chemotherapeutic drugs to MDR cells in vitro as well as inexperimental tumors in vivo (6). Clinical trials with MDR modulatorshave also shown some response in tumors that were otherwisenon-responsive to chemotherapy (7).

While constitutive P-gp expression in normal healthy tissues is believedto be an important protective mechanism against potentially toxicxenobiotics, during disease states, such as cancer, P-gp is recognizedas a major barrier to the bioavailability of administered drugs andthus, resistance to chemotherapy remains an obstacle to the successfultreatment of certain cancers (Johnstone et al. (2000), Ho et al.(2003)). Recent chemotherapeutic strategies have integrated the use ofhammerhead ribozymes against the MDRI gene (encodes for P-gp) and MDRItargeted anti-sense oligonucleotides (Fojo et al, 2003). Yet, despitethese advances, and MDR inhibitors in development that have progressedto the stage of clinical trials have been generally ineffective or onlyeffective at highly toxic doses (Baird et al., 2003). In addition, manyof these modulators adversely influence the pharmacokinetics andbio-distribution of co-administered chemotherapeutic drugs. Moreover,although siRNA mediated silencing of P-gp is a promising approach, thismethod may genetically alter cell fate and require delicate constructeddelivery systems that has, thus far, hampered its clinic usage.

Despite advances in the field, all MDR inhibitors in development thathave progressed to the stage of clinical trials have been widelyineffective or only effective at highly toxic doses (8). Furthermore,since most of the prior art modulators adversely influence thepharmacokinetics and biodistribution of co-administered chemotherapeuticdrugs, there remains a need for new, effective MDR and/or P-gpmodulators without the undesired side effects (4).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C. The S. Typhutmrium effector protein SipA modulates theexpression of P-gp by an extracellular effect (FIG. 1A) HCT8 intestinalepithelial cell monolayers were left untreated (−) or infected with wildtype (WT) S. Typhimurium SL1344 or SL1344 type III secretion systemtranslocon mutant strains (ΔsipB or ΔsipC) for 5 h. Whole cell lysateswere normalized for protein levels and probed for P-gp. GAPDH probingserved as a loading control. (FIG. 1B) HCT8 cells were infected withwild type SL1344 or an SL1344 SPI-1 deficient mutant strain, or exposedto wild-type SL1344-derived secreted protein extracts for 5 h, and thenprobed as in (FIG. 1A). (FIG. 1C) HCT8 cells were infected withwild-type SL1344, SL1344ΔsopA or ΔsipA, or SL1344ΔSipA complemented witha vector expressing SipA (ΔSipA/pSipA) for 5 h, and then probed as in(FIG. 1A). Densitometry was analyzed by ImageJ and presented as relativeto the untreated cells.

FIG. 2A-C. SipA down-regulates P-gp expression in a dose-dependentmanner. (FIG. 2A) HCT-8 cell monolayers were left untreated (−) orinfected with wild type SL1344 or exposed to 80 μg/ml or 160 μg/ml ofpurified SipA over a time course of 3 h. Normalized whole cell lysateswere then probed for P-gp and GAPDH. (FIG. 2B) HCT8 cell monolayers wereinfected with wild type SL1344 or exposed to purified lipopolysaccharide(LPS) from S. typhimurium (0.1 to 100 μg/ml) for 3 h, and then probed asin (FIG. 2A). (FIG. 2C) HCT8 cell monolayers were exposed to secretedprotein extracts from SL1344 wild type, ΔsipA or ΔSipA/pSipA for 3 h,and then probed as in (FIG. 2A).

FIG. 3A-B. SipA-induced, dose-dependent P-gp down-regulation isconserved in other cancer cell types. (FIG. 3A) MCF-7 breastadenocarcinoma cells were left untreated (−) or exposed to 80 μg/ml or160 μg/ml of purified SipA for 3 h. Normalized whole cell lysates werethen probed for P-gp and GAPDH. Densitometry was analyzed by ImageJ andpresented as relative to the untreated cells. (FIG. 3B) UM-UC-3 humanbladder carcinoma cells were left untreated or exposed to 80 μg/ml or160 μg/ml of purified SipA for 3 h, and then probed and analyzed as in(FIG. 3A).

FIG. 4A-D. S. Typhimurium modulates P-gp expression through acaspase-3-dependent mechanism. (FIG. 4A) HCT8 cell monolayers were leftuntreated (−) or infected with wild type SL1344 in the presence orabsence of pharmacological inhibitors of CASP-3 or CASP-1 (negativecontrol) for 5 h. Normalized whole cell lysates were then probed forP-gp and GAPDH. (FIG. 4B) HCT8 cell monolayers transfected with anonspecific siRNA vector control or with siRNA aimed at decreasingCASP-3 expression were left untreated or infected with wild type SL1344.Whole cell lysates were then probed as in (FIG. 4A). (FIG. 4C)Three-dimensional structure of mouse P-gp (PDB ID, 3G5U) depicted ascartoon and transparent surface: The cytoplasmic Caspase-3 cleavage site(⁴⁵⁴DGQD⁴⁵⁷) is shown in red. The putative CASP3 site ¹⁶⁴DVHD¹⁶⁷ is notshown. Numbers refer to the position of the amino acids in the proteinsequence. (FIG. 4D) HCT8 cell monolayers were infected with wild typeSL1344 for 1, 3 or 5 h, and then probed using a P-gp antibody capable ofdetecting P-gp cleavage products. Progressive P-gp modulation wasaccompanied by the occurrence of 90 and 60 kDs cleavage products.

FIG. 5A-B. SipA-AuNPs decrease the expression of P-gp at a SipA dosenearly 500 times lower than free SipA. (FIG. 5A) Schematic presentationof P-gp knockdown mechanism via SipA-AuNP. (FIG. 5B) HCT8 cellmonolayers were left untreated (−), exposed to 320 μg/ml 160 μg/ml ofpurified SipA, AuNP alone or AuNP-SipA (0.72 μg/ml of SipA).

FIG. 6A-D. The combined effects of SipA-AuNPs and exogenous doxorubicinprevent tumor growth. (FIG. 6A) Balb/c mice bearing subcutaneous CT26tumors (mean tumor volumes of approximately 0.5 mm³) received IPtreatments for 15 days as described in the Materials and Methodssection, (▪) Untreated, (●) AuNP alone, (▴A) SipA-AuNPs, (♦) Doxorubicinor (□) SipA-AuNP plus Doxorubicin (DOX). SipA-AuNPs conjugates improvethe efficacy of doxorubicin. (*P<0.0001). (FIG. 6B) Accumulation of goldnanoparticles in the tumors shown in (FIG. 6A) was evaluated by SEM andX-ray microanalysis. Color intensity represents tumor penetration. Thesections of tumor were imaged for X-ray analysis and X-ray mapping asdescribed in the Materials and Methods section. (FIG. 6C) P-gpexpression in the tumors shown in (FIG. 6A) was evaluated by westernblot. Tumors were homogenized and lysed. Whole cell lysates werenormalized for protein levels and probed for P-gp. Levels of P-gp werequantified by densitometry and presented on the bar cart. Densitometrywas performed using ImageJ and results are presented as relative to theuntreated cells. (FIG. 6D) Balb/c mice were infected with 10⁷ CFU ofeither SL1344 ΔSipA or SL1344 ΔSipA complemented with SipA (ΔSipA/pSipA)for 48 hours, after which the proximal colon was dissected, homogenizedand lysed. Whole cell lysates were normalized for protein levels andprobed for P-gp. Levels of P-gp were quantified by densitometry andpresented on the bar graph. Densitometry was performed using ImageJ andresults are presented as relative to the untreated cells. *P<0.0001(n=6).

FIG. 7: SipA exemplary amino acid sequence SEQ ID NO:01 of Salmonellaenterica subsp. enterica serovar Typhimurium str. SL1344 (GenBank:AAA86618.1) encoded by the DNA sequence (Locus taq) SL1344_2861 of theSalmonella enterica subsp. enterica serovar Typhimurium str. SL1344,complete genome sequence (NCBI Reference Sequence: NC_016810.1).

FIG. 8: Scheme of synthesis of the dithiolated tetra (ethylene glycol)carboxylic acid.

FIG. 9: The extracted ion chromatograph (EIC) peaks for peptideIPEPAAGPVPDGGK from the Sip A and SipA-AuNP samples.

FIG. 10A-B: Exemplary Homo sapiens PERP amino acid sequence SEQ ID NO:02(FIG. 10A) encoded by nucleotide sequence SEQ ID NO:03 (FIG. 10B) (NCBIReference Sequence: NM_22121.4).

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

“PERP,” “p53 apoptosis effector related to PMP-22” and “TP53 apoptosiseffector” interchangeably refer to a tetraspan membrane proteinoriginally identified as a transcriptional target of the p53 tumorsuppressor (Davies et al., PERP expression stabilizes active p53 viamodulation of p53-MDM2 interaction in is veal melanoma cells. Cell DeathBis 2, e136 (2011). Human PERP is exemplified by Homo sapiens PERP aminoacid sequence SEQ ID NO:02 (FIG. 10A) encoded by nucleotide sequence SEQID NO:03 (FIG. 10B) (NCBI Reference Sequence: NM_022121.4).

“P-glycoprotein,” “P-gp,” “MDRI protein” are used interchangeably torefer to a membrane transport protein that promotes the expulsion ofxenobiotics, which is a 170-kDa adenosine triphosphate (ATP)-dependentmultispecific drug transporter. P-gp is encoded by MDRI, and is amultidrug resistance ATP-binding cassette (ABC) membrane transporterresponsible for one aspect of the multi-drug resistance (MDR) phenotypein cancer cells (Krishna et al., Curr Med Chem Anticancer Agents 1, 163(August, 2001)). P-gp is exemplified by Homo sapiens (human) ABCB1ATP-binding cassette, sub-family B (MDR/TAP), member 1, Gene ID: 5243.Several reports have linked the overexpression of P-gp to adversetreatment outcomes in many cancers, thereby identifying this MDRphenotype as an important biologic target for pharmacologic modulation(Krishna et al, Curr Med Chem Anticancer Agents 163 (August 2001);Juliano et at, Biochmica et biophysica acta 455, 152 (Nov. 11, 1976)).Normal healthy tissues display baseline expression of P-gp, and it isbelieved to be an important protective mechanism against potentiallytoxic xenobiotics and to keep homeostasis. It is highly expressed inimportant pharmacological barriers, such as, placenta, brush bordermembrane of intestinal cells, the biliar canalicular membrane ofhepatocytes, the lumenal membrane in proximal tubules of kidneys, andthe epithelium that contributes to the blood-brain barrier (Gottesman,M. et al, Nat Rev Cancer, 2002 January; 2(1):48-58). Additionally, P-gpis expressed across different blood cells (Van de Ven,R. et al., JLeukoc Biol. 2009 November; 86(5):1075-87).

“Un-cleaved P-gp” refers to P-gp that has not been cleaved by caspase-3(CASP3) to produce cleavage products that comprise an approximately 90kDa P-gp cleavage product and/or an approximately 60 kDa P-gp cleavageproduct. Example 4 shows exemplary methods for determining the level ofun-cleaved P-gp.

“Protein 53,” “p53,” “tumor protein 53” and “TP53” are interchangeablyused to refer to a tumor suppressor protein that in humans is encoded bythe TP53 gene.

A “variant” or “homolog” of a polypeptide sequence of interest ornucleotide sequence of interest, refers to a sequence that has at least80% identity, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identitywith the polypeptide sequence of interest or nucleotide sequence ofinterest, respectively.

In one preferred embodiment, the variant has at least 95% identity tothe sequence of interest, including 95%, 96%, 97%, 98%, 99%, and 100%identity with the sequence of interest.

“Identity” when in reference to 2 or more sequences (e.g., DNA, RNA,and/or protein sequences) refers to the degree of similarity between the2 or more sequences, and is generally expressed as a percentage.Identity in amino acid or nucleotide sequences can be determined usingKarlin and Altschul's BLAST algorithm (Proc. Natl. Acad. Sci. USA, 1990,87, 2264-2268; Karlin, S. & Altschul, S F., Proc. Natl. Acad. Sci. USA,1993, 90, 5873). Programs called BLASTN and BLASTX have been developedusing the BLAST algorithm as a base (Altschul, S F. et al., J. Mol.Biol., 1990, 215, 403). When using BLASTN to analyze nucleotidesequences, the parameters can be set at, for example, score=100 and wordlength=12. In addition, when using BLASTX to analyze amino acidsequences, the parameters can be set at, for example, score=50 and wordlength=3. When using BLAST and the Gapped BLAST program, the defaultparameters for each program are used. Specific techniques for theseanalysis methods are the well known, e.g., on the website of theNational Center for Biotechnology Information.

“Purify” and grammatical equivalents thereof when in reference to adesirable component (such as cell, protein, nucleic acid sequence,carbohydrate, etc.) refer to the reduction in the amount of at least oneundesirable component (such as cell, protein, nucleic acid sequence,carbohydrate, sialic acid-glycoprotein etc.) from a sample, including areduction by any numerical percentage of from 5% to 100%, such as, butnot limited to, from 10% to 100%, from 20% to 100%, from 30% to 100%,from 40% to 100%, from 50% to 100%, from 60% to 100%, from 70% to 100%,from 80% to 100%, and from 90% to 100%. Thus purification results in“enrichment” (i.e., an increase) in the amount of the desirablecomponent relative to one or more undesirable components, resulting in amore concentrated form (relative to the starting material, such as thecell lysate and/or extracellular solution) of the desirable component.

“Cytotoxic” molecule refers any molecule that reduces proliferationand/or viability of a target cell, preferably, though not necessarily,killing the target cell. In a preferred embodiment, the cytotoxicmolecule is an anti-cancer toxin.

“Anti-cancer toxin” and “anti-cancer cytotoxin” is a molecule thatreduces proliferation of cancer cells and/or reduces viability of cancercells and/or reduces tumor size and/or reduces tumor number and/orreduces metastasis and/or increases apoptosis of cancer cells. Inpreferred embodiments, anti-cancer toxins delay the onset of developmentof tumor development and/or reduce the number, weight, volume, and/orgrowth rate of tumors. Cytotoxins are exemplified by, withoutlimitation, second messengers such as cAMP; Bacterial toxins such as theexemplary Pertussis toxin, Cholera toxin, and C3 exoenzyme; Lectins suchas Ricin A (Engert et al. Blood. 1997 Jan. 15; 89(2):403-10). Alsoincluded are toxins exemplified by Topoisomerase inhibitor such asetoposide, Campothecin irinotecan, topotecan, anthracyclines(doxorubicine, daunorubicine); Microtubule inhibitors such asvincristine, vinblastine, vinorelbine, paclitaxel, docetaxel; Platinumcontaining compounds such as cisplatin, carboplatin, oxaloplatin, etc.;Alkylating agents such as cyclophosphamide, and ifosfamide;Antimetabolites such as methotrexate and mercaptoprine; Anti-estrogenssuch as tamoxifen and toremifene; Retinoids such as all trans-retinoicacid; and others such as Adriamycin, gemcitabine, and 5-fluoruracil.

A number of the above-mentioned toxins also have a wide variety ofanalogues and derivatives, including, but not limited to, cisplatin,cyclophosphamide, misonidazole, tiripazamine, nitrosourea,mercaptopurine, methotrexate, flurouracil, epirubicin, doxorubicin,vindesine and etoposide. Analogues and derivatives include(CPA).sub.2Pt(DOLYM) and (DACH)Pt(DOLYM) cisplatin,Cis-(PtCL.sub.2(4,7-H-5-methyl-7-oxo-)1,2,4(triazolo(1,5-a)pyrimidine).sub.2),(Pt(cis-1,4-DACH)(trans-Cl.sub.2)(CBDCA)).multidot.-1/2MeOH cisplatin,4-pyridoxate diammine hydroxy platinum, Pt(II).Pt(II)(Pt.sub.2(NHCHN(C(CH.sub.2)(CH.s-ub.3))).sub.4), 254-S cisplatinanalogue, O-phenylenediamine ligand bearing cisplatin analogues, trans,cis-(Pt(OAc).sub.21.sub.2(en)), estrogenic 1,2-diarylethylenediamineligand (with sulfur-containing amino acids and glutathione) bearingcisplatin analogues, cis-1,4-diaminocyclohexane cisplatin analogues, 5′orientational isomer of cis-(Pt(NH.sub.3)(4-aminoTEMP-O){d(GpG)}),chelating diamine-bearing cisplatin analogues, 1,2-diarylethyleneamineligand-bearing cisplatin analogues, (ethylenediamine)platinum-(II)complexes, CI-973 cisplatin analogue, cis-diamminedichloroplatinum(II)and its analoguescis-1,1-cyclobutanedicarbosylato(2R)-2-methyl-1,4-butanediam-mineplatinum-(II)and cis-diammine(glycolato)platinum,cis-amine-cyclohexylamine-dichloroplatinum(II), gem-diphosphonatecisplatin analogues (FR 2683529),(meso-1,2-bis(2,6-dichloro-4-hydroxyplenyl)ethylenediamine)dichloroplatinum(II), cisplatin analogues containing a tethered dansylgroup, platinum(II) polyamines,cis-(3H)dichloro(ethylenediamine)platinu-m(II),trans-diamminedichloroplatinum(II) andcis-(Pt(NH.sub.3).sub.2(N.sub.3-cy-tosine)Cl),3H-cis-1,2-diaminocyclohexanedichloroplatinum(II) and3H-cis-1,2-diaminocyclohexane-malonatoplatinum (II),diaminocarboxylatoplatinum (EPA 296321),trans-(D,1)-1,2-diaminocyclohexane carrier ligand-bearing platinumanalogues, aminoalkylammoanthraqumone-derived cisplatin analogues,spiroplatin, carboplatin, iproplatin and JM40 platinum analogues,bidentate tertiary diamine-containing cisplatinum derivatives,platinum(II), platinum(IV), cis-diammine(1,1-cyclobutanedicarboxylato-)platinum(II) (carboplatin, JM8) andethylenediammine-malonatoplatinum(II) (JM40), JM8 and JM9 cisplatinanalogues, (NPr4)2((PtCL4).cis-(PtCl2-(NH2Me)2)), aliphatictricarboxylic acid platinum complexes (EPA 185225), cis-dichloro(aminoacid)(tert-butylamine)platinum-(II) complexes;4-hydroperoxycylcophosphamide, acyclouridine cyclophosphamidederivatives, 1,3,2-dioxa- and -oxazaphosphorinane cyclophosphamideanalogues, C5-substituted cyclophosphamide analogues, tetrahydrooxazinecyclophosphamide analogues, phenyl ketone cyclophosphamide analogues,phenylkelophosphamide cyclophosphamide analogues, ASTA Z-7557cyclophosphamide analogues,3-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)cy-clophosphamide,2-oxobis(2-β-chloroethylamino)-4-,6-dimethyl-1,3,2-oxazaphosphorinanecyclophosphamide, 5-fluoro- and 5-chlorocyclophosphamide, cis- andtrans-4-phenylcyclophosphamide, 5-bromocyclophosphamide,3,5-dehydrocyclophosphamide, 4-ethoxycarbonyl cyclophosphamideanalogues, arylaminotetrahydro-2H-1,3,2-oxazaphosphorine 2-oxidecyclophosphamide analogues, NSC-26271 cyclophosphamide analogues, benzoannulated cyclophosphamide analogues, 6-trifluoromethylcyclophosphamide,4-methylcyclophosphamide and 6-methycyclophosphamide analogues; FCE23762 doxorubicin derivative, annamycin, ruboxyl, anthracyclinedisaccharide doxorubicin analogue, N-(trifluoroacetyl)doxorubicin and4′-O-acetyl-N-(trifluoroacetyl)-doxorubicin, 2-pyrrolmodoxorubicin,disaccharide doxorubicin analogues,4-demethoxy-7-O-(2,6-dideoxy-4-O-(2,3,6-trideoxy-3-amino-α-L-lyxo-h-exopyranosyl)-α-L-lyxo-hexopyranosyl)adriamicinone doxorubicin disaccharide analog, 2-pyrrolinodoxorubicin,morpholinyl doxorubicin analogues, enaminomalonyl-β-alanine doxorubicinderivatives, cephalosporin doxorubicin derivatives, hydroxyrubicin,methoxymorpholino doxorubicin derivative, (6-maleimidocaproyl)hydrazonedoxorubicin derivative, N-(5,5-diacetoxypent-1-yl) doxorubicin, FCE23762 methoxymorpholinyl doxorubicin derivative, N-hydroxysuccinimideester doxorubicin derivatives, polydeoxynucleotide doxorubicinderivatives, morpholinyl doxorubicin derivatives (EPA 434960),mitoxantrone doxorubicin analogue, AD 198 doxorubicin analogue,4-demethoxy-3′-N-trifluoroacetyldoxorubicin, 4′-epidoxorubicin,alkylating cyanomorpholino doxorubicin derivative,deoxydihydroiodooxorubicin (EPA 275966), adriblastin,4′-deoxydoxorubicin, 4-demethyoxy-4′-o-methyldoxorubicin,3′-deamino-3′-hydroxydoxorubicin, 4-demethyoxy doxorubicin analogues,N-L-leucyl doxorubicin derivatives,3′-deamino-3′-(4-methoxy-1-piperidinyl) doxorubicin derivatives(4,314,054), 3′-deamino-3′-(4-mortholinyl) doxorubicin derivatives(4,301,277), 4-deoxydoxorubicin and 4′-o-methyldoxorubicin, aglyconedoxorubicin derivatives, SM 5887,MX-2,4′-deoxy-13(S)-dihydro-4′-iododoxorubicin (EP 275966), morpholinyldoxorubicin derivatives (EPA 434960),3′-deamino-3′-(4-methoxy-1-piperidi-nyl) doxorubicin derivatives(4,314,054), doxorubicin-14-valerate,morpholinodoxorubicin(5,004,606),3′-deamino-3′-(3′-cyano-4″-morpholinyldoxorubicin; 3′-deamino-3′-(3″-cyano-4″-morpholinyl)-13-dihydoxorubicin;(3′-deamino-3′-(3″-cyano-4″-morpholinyl) daunorabicin;3′-deamino-3′-(3″-cyano-4″-morpholinyl)-3-dihydrodaunorubicin; and3′-deamino-3′-(4″-morpholinyl-5-iminodoxorubicin and derivatives(4,585,859), 3′-deamino-3′-(4-methoxy-1-piperidinyl) doxorubicinderivatives (4,314,054) and 3-deamino-3-(4-morpholinyl) doxorubicinderivatives (4,301,277); 4,5-dimethylmisonidazole, azo and azoxymisonidazole derivatives; RB90740; 6-bromo and6-chloro-2,3-dihydro-1,4-benzothi-azines nitrosourea derivatives,diamine acid nitrosourea derivatives, amino acid nitrosoureaderivatives, 3′,4′-didemethoxy-3′,4′-dio-xo-4-deoxypodophyllotoxinnitrosourea derivatives, ACNU, tertiary phosphine oxide nitrosoureaderivatives, sulfamerizine and sulfamethizole nitrosourea derivatives,thymidine nitrosourea analogues, 1,3-bis(2-chloroethyl)-1-nitrosourea,2,2,6,6-tetramethyl-1-oxopiperidiunium nitrosourea derivatives (U.S.S.R.1261253), 2- and 4-deoxy sugar nitrosourea derivatives (4,902,791),nitroxyl nitrosourea derivatives (U.S.S.R. 1336489), fotemustine,pyrimidine (II) nitrosourea derivatives, CGP 6809, B-3839,5-halogenocytosine nitrosourea derivatives,1-(2-chloroethyl)-3-isobu-tyl-3-(β-maltosyl)-1-nitrosourea,sulfur-containing nitrosoureas, sucrose,6-((((2-chloroethyl)nitrosoamino-)carbonyl)amino)-6-deoxysucrose (NS-1C)and 6′-((((2-chloroethyl)nitrosoamino)carbonyl)amino)-6′-deoxysucrose(NS-1D) nitrosourea derivatives, CHCC, RFCNU and chlorozotocin, CNUA,1-(2-chloroethyl)-3-isobutyl-3-(β-maltosyl)-1-nitrosourea, choline-likenitrosoalkylureas, sucrose nitrosourea derivatives (JP 84219300), sulfadrug nitrosourea analogues, DONU, N,N′-bis(N-(2-chloroethyl)-N-nitrosocarbamoyl)cystamine (CNCC),dimethylnitrosourea, GANU, CCNU, 5-aminomethyl-2′-deoxyuridinenitrosourea analogues, TA-077, gentianose nitrosourea derivatives (JP 8280396), CNCC, RFCNU, RPCNU AND chlorozotocin (CZT), thiocolchicinenitrosourea analogues, 2-chloroethyl-nitrosourea, ACNU,(1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosoureahydrochloride), N-deacetylmethyl thiocoichicine nitrosourea analogues,pyridine and piperidine nitrosourea derivatives, methyl-CCNU,phensuzimide nitrosourea derivatives, ergoline nitrosourea derivatives,glucopyranose nitrosourea derivatives (JP 78 95917),1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea,4-(3-(2-chloroethyl)-3-nitrosoureid-o)-cis-cyclohexanecarboxylic acid,RPCNU (ICIG 1163), IOB-252, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU),1-tetrahydroxycyclopentyl-3-nitroso-3-(2-chloroethyl)-urea (4,039,578),d-1-1-(β-chloroethyl)-3-(2-oxo-3-hexahydroazepinyl)-1-nitrosourea(3,859,277) and gentianose nitrosourea derivatives (JP 57080396);6-S-aminoacyloxymethyl mercaptopurine derivatives, 6-mercaptopurine(6-MP), 7,8-polymethyleneimidazo-1,3,2-diazaph-osphorines, azathioprine,methyl-D-glucopyranoside mercaptopurine derivatives and s-alkynylmercaptopurine derivatives; indoline ring and a modified ornithine orglutamic acid-bearing methotrexate derivatives, alkyl-substitutedbenzene ring C bearing methotrexate derivatives, benzoxazine orbenzothiazine moiety-bearing methotrexate derivatives,10-deazaaminopterin analogues, 5-deazaaminopterin and5,10-dideazaaminopterin methotrexate analogues, indoline moiety-bearingmethotrexate derivatives, lipophilic amide methotrexate derivatives,L-threo-(2S,4S)-4-fluoro-glutamic acid and DL-3,3-difluoroglutamicacid-containing methotrexate analogues, methotrexatetetrahydroquinazoline analogue, N-(ac-aminoacyl) methotrexatederivatives, biotin methotrexate derivatives, D-glutamic acid orD-erythrou, threo-4-fluoroglutamic acid methotrexate analogues,β,γ-methano methotrexate analogues, 10-deazaaminopterin (10-EDAM)analogue, γ-tetrazole methotrexate analogue, N-(L-α-aminoacyl)methotrexate derivatives, meta and ortho isomers of aminopterin,hydroxymethylmethotrexate (DE 267495), γ-fluoromethotrexate,polyglutamyl methotrexate derivatives, gem-diphosphonate methotrexateanalogues (WO 88/06158), α- and γ-substituted methotrexate analogues,5-methyl-5-deaza methotrexate analogues (4,725,687), N.delta.-acyl-Nα-(4-amino-4-deoxypteroyl)-L-ornithine derivatives, 8-deaza methotrexateanalogues, acivicin methotrexate analogue, polymeric platinolmethotrexate derivative,methotrexate-γ-dimyristoylphophatidylethanolamine, methotrexatepolyglutamate analogues, poly-γ-glutamyl methotrexate derivatives,deoxyuridylate methotrexate derivatives, iodoacetyl lysine methotrexateanalogue, 2.omega.-diaminoalkanoid acid-containing methotrexateanalogues, polyglutamate methotrexate derivatives, 5-methyl-5-deazaanalogues, quinazoline methotrexate analogue, pyrazine methotrexateanalogue, cysteic acid and homocysteic acid methotrexate analogues(4,490,529), γ-tert-butyl methotrexate esters, fluorinated methotrexateanalogues, folate methotrexate analogue, phosphonoglutamic acidanalogues, poly (L-lysine) methotrexate conjugates, dilysine andtrilysine methotrexate derivates, 7-hydroxymethotrexate, poly-γ-glutamylmethotrexate analogues, 3′,5′-dichloromethotrexate, diazoketone andchloromethylketone methotrexate analogues, 10-propargylaminopterin andalkyl methotrexate homologs, lectin derivatives of methotrexate,polyglutamate methotrexate derivatives, halogentated methotrexatederivatives, 8-alkyl-7,8-dihydro analogues, 7-methyl methotrexatederivatives and dichloromethotrexate, lipophilic methotrexatederivatives and 3′, 5′-dichloromethotrexate, deaza amethopterinanalogues, MX068 and cysteic acid and homocysteic acid methotrexateanalogues (EPA 0142220); N3-alkylated analogues of 5-fluorouracil,5-flourouracil derivatives with 1,4-oxaheteroepane moieties,5-fluorouracil and nucleoside analogues, cis- andtrans-5-fluoro-5,6-dihydro-6-alkoxyuracil, cyclopentane 5-flourouracilanalogues, A-OT-fluorouracil,N4-trimethoxybenzoyl-5′-deoxy-5-fluoro-cytidine and5′-deoxy-5-fluorouridine, 1-hexylcarbamoyl-5-fluorouracil, B-3839,uracil-1-(2-tetrahydrofuryl)-5-flourouacil,1-(2′-deoxy-2′-fluoro-βD-arabinofuranosyl)-5-fl-uorouracil,doxifluridine, 5′-deoxy-5-fluorouridine,1-acetyl-3-O-toluyl-5-fluorouracil,5-fluorouracil-m-formylbenzene-sulfonate (JP 55059173),N′-(2-furanidyl)-5-flourouracil (JP 53149985) and1-(2-tetrahydrofuryl)-5-fluorouracil (JP 52089680); 4′-epidoxorubicin;N-substituted deacetylvinblastine amide (vindesine) sulfates; andCu(II)-VP-16 (etoposide) complex, pyrrolecarboxamidino-bearing etoposideanalogues, 40-amino etoposide analogues, γ-lactone ring-modifiedarylamino etoposide analogues, N-glucosyl etoposide analogue, etoposideA-ring analogues, 4′-deshydroxy-4′-methyl etoposide, pendulum ringetoposide analogues and E-ring desoxy etoposide analogues.

“Nanoparticle” refers to a particle of a solid (such as a metal,polymer, oxide, etc.) having one or more dimensions of approximately 100nm or less. Enablement: generic methods for “linking” (i.e.,“conjugating”) molecules (such as chemotherapeutic agent, antibioticagent, antifungal agent, antiparasitic agent, antiviral agent, SipA.etc.) to nanoparticles for drug delivery to tissue (such as cancertissue), are known in the art (e.g., U.S. Pat. Nos. 8,318,208,8,318,211, 8,246,968, 8,193,334, 8,063,131, 7,727,554, 7,563,457,7,550,441, 7,550,282, 7,387,790, 7,348,030, 5,718,919, 5,503,723,5,429,824; U.S. Patent Publication No. US 2012/0302516, WO 2008/151049)including gold nanoparticles (e.g., U.S. Pat. No. 8,323,694). Exemplarymethods for conjugating SipA to gold nanoparticles are described hereinin Examples 1, 5 and 7.

“Operably conjugated” and “operably linked” when in reference to thelinkage between two molecules, such as the linkage between ananoparticle and another molecules (such as SipA, cytotoxin,chemotherapeutic agent, antibiotic agent, antifungal agent,antiparasitic agent, antiviral agent, etc.) means that the molecules arelinked such that each molecule performs its intended and/or biologicalfunction (e.g., SipA reduces the level of expression of P-gp in cellsand/or reduces the level of functional un-cleaved P-gp in cells and/orincreases the level of expression of PEEP, etc.). Linkage may be direct,indirect, non-covalent, covalent, etc. In a preferred embodiment,linkage between nanoparticles and proteins is covalent to reduce proteindissociation or aggregation.

The terms “specifically binds” and “specific binding” when made inreference to the binding of two molecules (e.g. antibody to an antigen),arc., refer to an interaction of the two molecules that is dependentupon the presence of a particular structure on one or both of themolecules. For example, if an antibody is specific for epitope “A” onthe molecule, then the presence of a protein containing epitope A (orfree, unlabeled A) in a reaction containing labeled “A” and the antibodywill reduce the amount of labeled A bound to the antibody.

“Antibody” refers to an immunoglobulin (e.g., IgG, IgM, IgA, IgE, IgD,etc.).

“Antigen-binding portion” of an antibody refers to a fragment of theantibody that specifically binds to an antigen. “Antigen-bindingportion” includes a “Variable domain” (also referred to as the “Fvregion”) for binding to antigens. More specifically, variable loops,three each on the light (V_(L)) and heavy (V_(H)) chains are responsiblefor binding to the antigen. These loops are referred to as the“complementarity determining regions” (“CDRs”) and “idiotypes.”“Antigen-binding portion” includes the Fab region, F(ab′)2 fragment,pFc′ fragment, and Fab′ fragments. The “Fab region” and “fragment,antigen binding region,” interchangeably refer to portion of theantibody arms of the immunoglobulin “Y” that function in bindingantigen. The Fab region is composed of one constant and one variabledomain from each heavy and light chain of the antibody. Methods areknown in the art for the construction of Fab expression libraries (Huseet al., Science, 246:1275-1281 (1989)) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.In another embodiment, Fc and Fab fragments can be generated by usingthe enzyme papain to cleave an immunoglobulin monomer into two Fabfragments and an Fc fragment. The enzyme pepsin cleaves below the hingeregion, so a “F(ab′)2 fragment” and a “pFc′ fragment” is formed. TheF(ab′)2 fragment can be split into two “Fab′ fragments” by mildreduction.

The “Fc” and “Fragment, crystallizable” region interchangeably refer toportion of the base of the immunoglobulin “Y” that function in role inmodulating immune cell activity. The Fc region is composed of two heavychains that contribute two or three constant domains depending on theclass of the antibody. By binding to specific proteins, the Fc regionensures that each antibody generates an appropriate immune response fora given antigen. The Fc region also binds to various cell receptors,such as Fc receptors, and other immune molecules, such as complementproteins. By doing this, it mediates different physiological effectsincluding opsonization, cell lysis, and degranulation of mast cells,basophils and eosinophils. In an experimental setting, Fc and Fabfragments can be generated in the laboratory by cleaving animmunoglobulin monomer with the enzyme papain into two Fab fragments andan Fc fragment.

“Cyclic-arginine-glycine-aspartic acid,” “cRGD,” “cGRGDdvc” and “LXW7”interchangeably refer to an arginine-glycine-aspartic acid peptidecyclized by a disulfide bond and with a built-in handle at the carboxylterminus.

“Cancer cell” refers to a cell undergoing early, intermediate oradvanced stages of multi-step neoplastic progression as previouslydescribed (Pitot et al, Fundamentals of Oncology, 15-28 (1978)). Thisincludes cells in early, intermediate and advanced stages of neoplasticprogression including “pre-neoplastic cells (i.e., “hyperplastic cellsand dysplastic cells), and neoplastic cells in advanced stages ofneoplastic progression of a dysplastic cell. “Cancer” includes cellsthat may or may not be metastatic, and is exemplified by ovarian cancer,breast cancer, lung cancer, prostate cancer, cervical cancer, pancreaticcancer, colon cancer, stomach cancer, esophagus cancer, mouth cancer,tongue cancer, gum cancer, skin cancer (e.g., melanoma, basal cellcarcinoma, Kaposi's sarcoma, etc.), muscle cancer, heart cancer, livercancer, bronchial cancer, cartilage cancer, bone cancer, testis cancer,kidney cancer, endometrium cancer, uterus cancer, bladder cancer, bonemarrow cancer, lymphoma cancer, spleen cancer, thymus cancer, thyroidcancer, brain cancer, neuron cancer, mesothelioma, gall bladder cancer,ocular cancer (e.g., cancer of the cornea, cancer of uvea, cancer of thechoroids, cancer of the macula, vitreous humor cancer, etc.), jointcancer (such as synovium cancer), glioblastoma, lymphoma, and leukemia.In a particularly preferred embodiment, the cancer comprises one or moreof a colon cancer (see Example 2), colorectal cancer, gastrointestinalcancer, breast cancer (see Example 3), bladder cancer (see Example 3),kidney cancer, leukemia, brain cancer, sarcoma, astrocytoma, acutemyelogenous leukemia (AML), and diffuse large B-lymphoma.

“Symptom” is a sign of disease. Cancer symptoms include, but are notlimited to, weight loss, fever, fatigue, bleeding or discharge (lung,colon, rectal, cervix endometrium, bladder, kidney and/or breastcancers), sores that do not heal (skin and/or oral cancers), whitepatches inside the mouth or white spots on the tongue (leukoplakia inmouth cancer), thickening or lumps (breast testicle, and/or lymph nodecancers), tumor size, tumor rate of growth, indigestion or troubleswallowing (esophagus, stomach, and/or throat cancers), changes in sizeor color of moles (melanoma), cough or hoarseness (lung, voice boxand/or thyroid gland cancers). Multiple sclerosis symptoms include, butare not limited to, numbness or weakness in one or more limbs, partialor complete loss of central vision, usually in one eye, often with painduring eye movement (optic neuritis), double vision or blurring ofvision, tingling or pain in parts of the body, electric-shock sensationsthat occur with certain head movements, tremor, lack of coordination orunsteady gait, slurred speech, fatigue and/or dizziness. Autoimmunedisease symptoms include, but are not limited to, extreme fatigue,muscle and joint pain, muscle weakness, swollen glands, inflammation,susceptibility to infections, sleep disturbances, weight loss or gain,low blood sugar, blood pressure changes, Candida yeast infections,allergies, digestive problems such as abdominal pain, bloating,tenderness, heartburn, cramps, constipation, diarrhea and excessive gas(“leaky gut syndrome”), anxiety and depression, memory problems, thyroidproblems (hypothyroidism and/or hyperthyroidism) that can manifest aslow body temperature and excessive hair loss, re-current headaches, lowgrade fevers, and/or re-current miscarriage. Human ImmunodeficiencyVirus (HIV) infection symptoms include, but are not limited to, fatigue,diarrhea, nausea, vomiting, fever, chills, night sweats, muscle aches,sore throat, swollen lymph nodes, ulcers in the mouth, wasting syndromeat late stages, and/or opportunistic infections which occur in patientswith a damaged immune system.

“Non-cancerous cell” refers to a cell that is not a cancer cell, such asa cell that is not undergoing early, intermediate or advanced stages ofmulti-step neoplastic progression.

A cell that is “resistant to a cytotoxin” refers to a cell whose rate ofgrowth is not substantially reduced in the presence of the cytotoxin ascompared to in the absence of the cytotoxic

A “control” sample or cell refers to a sample or cell used for comparingto another sample or cell by maintaining the same conditions in thecontrol and other samples or cells, except in one or more particularvariable in order to infer a causal significance of this varied one ormore variable on a phenomenon. For example, a non-cancerous cell is acontrol cell vis-à-vis a cancer cell. In another example, a cell that isnot infected with a virus is a control cell vis-à-vis a cell that isinfected with the virus. Also, for example, a “positive control sample”is a control sample in which the phenomenon is expected to occur. Forexample, a “negative control sample” is a control sample in which thephenomenon is not expected to occur.

Cells that “overexpress” a protein and/or nucleotide sequence refer tocells that produce a higher level of the protein and/or nucleotidesequence compared to a control cell.

A “subject” Includes any multicellular animal, preferably a “mammal.”Mammalian subjects include humans, non-human primates, murines, ovines,bovines, ruminants, lagomorphs, porcines, caprines, equines, canines,felines, aves, etc.). Thus, mammalian subjects are exemplified by mouse,rat, guinea pig, hamster, ferret and chinchilla.

A subject “in need” of reducing one or more symptoms of a diseaseincludes a subject that exhibits and/or is at risk of exhibiting one ormore symptoms of the disease. For Example, subjects may be at risk basedon family history, genetic factors, environmental factors, etc. Thisterm includes animal models of the disease. Thus, administering acomposition (which reduces a disease and/or which reduces one or moresymptoms of a disease) to a subject in need of reducing the diseaseand/or of reducing one or more symptoms of the disease includesprophylactic administration of the composition (i.e., before the diseaseand/or one or more symptoms of the disease are detectable) and/ortherapeutic administration of the composition (i.e., after the diseaseand/or one or more symptoms of the disease are detectable).

A subject “at risk” for disease refers to a subject that is predisposedto contracting and/or expressing one or more symptoms of the disease.This predisposition may be genetic (e.g., a particular genetic tendencyto expressing one or more symptoms of the disease, such as heritabledisorders, etc.), or due to other factors (e.g., environmentalconditions, exposures to detrimental compounds, including carcinogens,present in the environment, etc.). The term subject “at risk” includessubjects “suffering from disease,” i.e., a subject that is experiencingone or more symptoms of the disease. It is not intended that the presentinvention be limited to any particular signs or symptoms. Thus, it isintended that the present invention encompass subjects that areexperiencing any range of disease, from sub-clinical symptoms tofull-blown disease, wherein the subject exhibits at least one of theindicia (e.g., signs and symptoms) associated with the disease.

The terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,” andgrammatical equivalents (including “lower,” “smaller,” etc.) when inreference to the level of any molecule (e.g., amino acid sequence suchas P-gp or PERP, and nucleic acid sequence such as a sequence encodingP-gp or PERP, antibody, etc.), cell, and/or phenomenon (e.g., level ofexpression of a gene such as the gene encoding P-gp or PERP, diseasesymptom, cell proliferation, cell viability, tumor size, tumor number,level of binding of two molecules, enzyme activity, biological activity,etc.) in a first sample (or in a first subject) relative to a secondsample (or relative to a second subject), mean that the quantity ofmolecule, cell and/or phenomenon in the first sample (or in the firstsubject) is lower than in the second sample (or in the second subject)by any amount that is statistically significant using any art-acceptedstatistical method of analysis. In one embodiment, the quantity ofmolecule, cell and/or phenomenon in the first sample (or in the firstsubject) is at least 10% lower than, at least 25% lower than, at least50% lower than, at least 75% lower than, and/or at least 90% lower thanthe quantity of the same molecule, cell and/or phenomenon in the secondsample (or in the second subject). In another embodiment, the quantityof molecule, cell, and/or phenomenon in the first sample (or in thefirst subject) is lower by any numerical percentage from 5% to 100%,such as, but not limited to, from 10% to 100%, from 20% to 100%, from30% to 100%, from 40% to 100%, from 50% to 100%, from 60% to 100%, from70% to 100%, from 80% to 100%, and from 90% to 100% lower than thequantity of the same molecule, cell and/or phenomenon in the secondsample (or in the second subject). In one embodiment, the first sample(or the first subject) is exemplified by, but not limited to, a sample(or subject) that has been manipulated using the invention'scompositions and/or methods. In a further embodiment the second sample(or the second subject) is exemplified by, but not limited to, a sample(or subject) that has not been manipulated using the invention'scompositions and/or methods. In an alternative embodiment, the secondsample (or the second subject) is exemplified by, but not limited to, asample (or subject) that has been manipulated, using the invention'scompositions and/or methods, at a different dosage and/or for adifferent duration and/or via a different route of administrationcompared to the first subject. In one embodiment, the first and secondsamples (or subjects) may be the same, such as where the effect ofdifferent regimens (e.g., of dosages, duration, route of administration,etc.) of the invention's compositions and/or methods is sought to bedetermined on one sample (or subject). In another embodiment, the firstand second samples (or subjects) may be different, such as whencomparing the effect of the invention's compositions and/or methods onone sample (subject), for example a patient participating in a clinicaltrial and another individual in a hospital.

The terms “increase,” “elevate,” “raise,” and grammatical equivalents(including “higher,” “greater,” etc.) when in reference to the level ofany molecule (e.g., amino acid sequence such as P-gp or PERP, andnucleic acid sequence such as a sequence encoding P-gp or PERP,antibody, etc.), cell, and/or phenomenon (e.g., level of expression of agene such as the gene encoding P-gp or PERP, disease symptom, cellproliferation, cell viability, tumor size, tumor number, level ofbinding of two molecules, enzyme activity, biological activity, etc.) ina first sample (or in a first subject) relative to a second sample (orrelative to a second subject), mean that the quantity of the molecule,cell and/or phenomenon in the first sample (or in the first subject) ishigher than in the second sample (or in the second subject) by anyamount that is statistically significant using any art-acceptedstatistical method of analysis. In one embodiment, the quantity of themolecule, cell and/or phenomenon in the first sample (or in the firstsubject) is at least 10% greater than, at least 25% greater than, atleast 50% greater than, at least 75% greater than, and/or at least 90%greater than the quantity of the same molecule, cell and/or phenomenonin the second sample (or in the second subject). This includes, withoutlimitation, a quantity of molecule, cell, and/or phenomenon in the firstsample (or in the first subject) that is at least 10% greater than, atleast 15% greater than, at least 20% greater than, at least 25% greaterthan, at least 30% greater than, at least 35% greater than, at least 40%greater than, at least 45% greater than, at least 50% greater than, atleast 55% greater than, at least 60% greater than, at least 65% greaterthan, at least 70% greater than, at least 75% greater than, at least 80%greater than, at least 85% greater than, at least 90% greater than,and/or at least 95% greater than the quantity of the same molecule, celland/or phenomenon in the second sample (or in the second subject). Inone embodiment, the first sample (or the first subject) is exemplifiedby, but not limited to, a sample (or subject) that has been manipulatedusing the invention's compositions and/or methods. In a furtherembodiment, the second sample (or the second subject) is exemplified by,but not limited to, a sample (or subject) that has not been manipulatedusing the invention's compositions and/or methods. In an alternativeembodiment, the second sample (or the second subject) is exemplified by,but not limited to, a sample (or subject) that has been manipulated,using the invention's compositions and/or methods, at a different dosageand/or for a different duration and/or via a different route ofadministration compared to the first subject. In one embodiment, thefirst and second samples (or subjects) may be the same, such as wherethe effect of different regimens (e.g., of dosages, duration, route ofadministration, etc,) of the Invention's compositions and/or methods issought to be determined on one sample (or subject). In anotherembodiment, the first and second samples (or subjects) may be different,such as when comparing the effect of the invention's compositions and/ormethods on one sample (subject), for example a patient participating ina clinical trial and another individual in a hospital.

The term “not substantially reduced” when in reference to the level ofany molecule (e.g., amino acid sequence such as P-gp or PERP, andnucleic acid sequence such as a sequence encoding P-gp or PERP,antibody, etc.), cell, and/or phenomenon (e.g., level of expression of agene such as the gene encoding P-gp or PERP, disease symptom, cellproliferation, cell viability, tumor size, tumor number, level ofbinding of two molecules, enzyme activity, biological activity, etc.) ina first sample (or in a first subject) relative to a second sample (orrelative to a second subject), means that the quantity of molecule, celland/or phenomenon in the first sample (or in the first subject) is from91% to 100% of the quantity in the second sample (or in the secondsubject).

The terms “alter” and “modify” when in reference to the level of anymolecule and/or phenomenon refer to an increase and/or decrease.

SUMMARY OF THE INVENTION

The invention provides a method for reducing one or more symptoms ofcancer in a mammalian subject in need thereof, comprising administeringto said subject a composition comprising purified SipA. In oneembodiment, said SipA is operably conjugated to a nanoparticle. Inanother embodiment, said cancer comprises cancer cells resistant to atleast one cytotoxin. In yet another embodiment, said cancer comprisescancer cells that overexpress one or more of P-gp and p53 compared to acontrol cell. In a further embodiment, the method optionally furthercomprises administering to said subject one or more cytotoxin. In oneembodiment, said SipA is administered in an amount that is effective inone or more of a) reducing the level of expression of P-gp in cells ofsaid cancer, b) reducing the level of un-cleaved P-gp in cells of saidcancer, and c) increasing the level of expression of PERP in cells ofsaid cancer. In yet another embodiment, said method further comprisesdetermining the level of expression of P-gp in cells of said cancer. Ina particular embodiment, said SipA is operably conjugated to acytotoxin. In another embodiment, said SipA is operably conjugated to atargeting agent that specifically binds to cells of said cancer. In analternative embodiment, said targeting agent comprises an antibody, oran antigen-binding portion thereof. In one preferred embodiment, saidtargeting agent comprises cyclic-arginine-glycine-aspartic acid (cRGD)peptide. In an alternative embodiment, said targeting agent comprisesfolic acid.

The invention further provides a method for reducing one or moresymptoms of a disease in a mammalian subject in need thereof, whereinsaid disease is associated with cells that overexpress one or more ofP-gp and p53, said method comprising administering to said subject acomposition comprising purified SipA, wherein said SipA is in an amountthat is effective in one or more of a) reducing the level of expressionof P-gp in said cells, b) reducing the level of un-cleaved P-gp in saidcells, and c) increasing the level of expression of PERP in said cells.In one embodiment, said disease is selected from the group consisting ofcancer, multiple sclerosis, autoimmune disease, and HumanImmunodeficiency Vims (HIV) infection.

Also provided by the invention is a method comprising administering to amammalian cell a composition comprising purified SipA, wherein said SipAis in an amount that is effective in one or more of a) reducing thelevel of expression of P-gp in said cell, b) reducing the level ofun-cleaved P-gp in said cell, and c) increasing the level of expressionof PERP in said cell. In one embodiment, said cell overexpresses one ormore of said P-gp and of p53 compared to a control cell. In anotherembodiment, said cell that overexpresses said P-gp is selected from thegroup consisting of cancer cell and non-cancerous cell. In oneembodiment, said cell is in vitro or in vivo. In yet another embodiment,said non-cancerous cell comprises a lymphocyte cell. In a furtherembodiment, said non-cancerous cell comprises an intestinal epithelialcell.

The invention further provides a nanoparticle comprising one or morepurified SipA.

The invention additionally provides a composition comprising any one ormore of the nanoparticles described herein, and at least onepharmaceutical acceptable diluent or excipient.

The invention also provides a method for increasing apoptosis of cancercells in a mammalian subject in need thereof, comprising administeringto said subject a composition comprising purified SipA.

BRIEF DESCRIPTION OF THE INVENTION

The invention provides the seminal discovery that a type III secretedeffector protein. SipA (Salmonella invasion protein A), isolated fromthe enteric pathogen Salmonella enterica serovar typhimurium has thecombined role of functionally down-regulating MDRI (or P-glycoprotein),and triggering pathways that stabilize active p53, ultimately drivingapoptotic responses.

The invention also provides the surprising discovery of a link between amicroorganism that is targeted specifically to tumors, and theregulation of multidrug resistance transporters. Data herein demonstratethat colonization of human colon cancer cell lines (that overexpressP-gp) by wild-type S. Typhimurium led to a profound functional decreaseand loss of protein expression in the multidrug resistance proteintransporter, P-gp (5).

In particular, the invention provides the discovery that SipA presents amajor advance with respect to previously developed small moleculeentities that target MDR and/or p53 drug-based strategies because it amolecule derived from a pathogenic microorganism evolutionary programmedto biologically engage epithelial cells and is also stable in hostilemicroenvironments, such as cancers.

The invention also provides the discovery that expression of P-gp andactivation of apoptosis (programmed cell death) share an inverserelationship. P-gp protein expression plays a major role in promotingcell survival, where it functions primarily as an anti-apoptoticmolecule presumably by pumping out enzymes critical to catalyzing theapoptotic cascade. Accordingly, by functionally down-regulating P-gp,the invention's nanoparticle possesses the additional advantage ofdriving tumors to become more sensitive to apoptosis. Improved treatmentthat targets apoptosis is based on two key observations: 1) Many of thechanges contributing to cancer development also diminish the ability ofcells to undergo apoptosis (9). When this death process is inhibited,damaged or defective cells that ordinarily would be eliminated insteadaccumulate and cause significant pathologic problems; and ii) a varietyof studies have demonstrated that apoptosis is a frequent outcome ofeffective therapy (9). Consequently, one of the invention's advantagesis to facilitate apoptosis in neoplastic cells.

The invention further provides the surprising and serendipitousdiscovery that the S. Typhimurium effector, SipA, promotes theproduction of PERP (p53 apoptosis effector related to—PMP-22) inepithelial cells, and that SipA enhances and stabilizes p53 activity.

Data herein demonstrate that SipA does not need to enter the epithelialcell cytosol to stimulate signal transduction pathways but, rather,functions extracellularly at the epithelial cell surface, where itengages a specific receptor. This finding is a paradigm-shiftingsurprising discovery since it challenges the long-held view that typeIII secretion system effector proteins must be directly delivered intohost cells from bacterial cells to engage signal transduction pathways.

The invention also provides a drug nanocarrier that addresses theshortcomings of traditional chemotherapeutic treatment, and that targetsmultidrug-resistant tumors while simultaneously stabilizing active p53,a tumor suppressor protein. The design of the novel chemotherapeutic andSipA co-conjugated drug delivery system capitalizes the unique chemicaland physical properties of the nanoparticle, biochemical functionalactivities of SipA, and pharmaceutical effectiveness of chemotherapeuticagents (such as the FDA approved doxorubicin). The invention'snanoparticle compositions establish a new paradigm in chemotherapeuticdrug delivery, as treatment methods using this nanocarrier offerunprecedented therapeutic potential by drastically improving efficacywhile minimizing drug associated side effects. The invention'scompositions and methods therefore will profoundly change the waydisease, and particularly cancer, is treated.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods for reducing one or moresymptoms of disease by administering compositions comprising apolypeptide sequence having at least 95% identity to SipA (SEQ IDNO:01). The invention's compositions and methods are particularlyadvantageous in reducing symptoms of diseases that are associated withoverexpression of P-gp and/or p53. The invention's compositions andmethods are useful in reducing cancer symptoms and/or cancer multidrugresistance (MDR). The invention is further described under (1)Salmonella enterica serovar Typhimurium (S. Typhimurium) interactionswith host cells, (2) Salmonella T3SS effector protein (SipA), (3)Methods for reducing disease symptoms, and (4) SipA conjugated tonanoparticles.

1. Salmonella enterica serovar Typhimurium (S. Typhimurium) Interactionswith Host Cells

Bacterial pathogens have been investigated as therapeutic agents fortumors for over 150 years (1). As an example, Salmonella entericaserovar Typhimurium (S. Typhimurium) is a facultative enteric pathogenthat causes food poisoning in humans resulting in gastroenteritis. Thispathogen can also selectively grow in tumors following systemicadministration and is able to modulate numerous biochemical pathwaysacross a broad spectrum of cell types (i.e., gut, kidney, lung,macrophages) (2, 3) (4). Therefore, the invention's compositions andmethods that harness these traits afford unique opportunities toovercome many of the delivery barriers that hinder conventionalchemotherapeutics.

S. typhimurium initiates infection and controls the fate of the hostcells by invading enterocytes predominantly located within the distalileum, and has evolved the use of a needle-like structure, known as thetype III secretion system to guide its pathogenesis (5). By way of thissophisticated secretion system, numerous Salmonella effector proteinsare secreted from the bacterium and then are translocated into thetarget cell cytosol. Such secreted effectors have high potential astherapeutic agents because they have co-evolved with the host and areextremely adept at interacting with host cell proteins involved in themodulation numerous signaling transduction pathways that are commontargets fundamental in the development of therapeutics of inflammatorydiseases and cancer (4, 5).

The inventors investigated Salmonella-host cell interactions with regardto the expression and functionality of P-gp. Recent reports have linkedthe overexpression of P-gp to adverse treatment outcomes in manycancers, thereby identifying this MDR phenotype as an important biologictarget for pharmacologic modulation (6, 7). The inventors' prior studiesrevealed that colonization of S. Typhimurium with human colon cancercell lines that overexpress P-gp leads to a profound functional decreaseand loss of protein expression in P-gp (8). There are also reportsdocumenting the ability of S. Typhimurium to target and selectively growin tumors (accumulating 2000-fold more in tumors than in other healthyorgans (3)).

2. Salmonella T3SS Effector Protein (SipA)

Data herein identify that the Salmonella type III secretion effector,SipA, is responsible for the effect of P-gp downregulation, and showthat the Salmonella Typhimurium secreted effector protein, SipA, canselectively and robustly down regulate P-gp. Data herein shows that SipAmodulates P-gp expression in several cancers that are known toover-express P-gp, such as colon, kidney, and breast cancer. Theinvention exploits this virulence determinant in the development of anovel strategy aimed at reducing (including reversing) multidrugresistance in tumors. Since SipA is a stable molecule that hasco-evolved with the human host, this virulence factor represents a majoradvance with respect to previously developed small molecule entitiesthat target MDR.

Exploiting these observations, the invention further provides atherapeutic application where the inventors engineered a SipA conjugatedgold nanoparticle (SipA-AuNP) system, which mimics the ability toreverse multidrug resistance. Using this system, the inventors foundthat a AuNP conjugated with SipA can reduce P-gp expression in cancercells at a SipA dose that is nearly 500 times lower than free unboundSipA. The inventors also demonstrate that the SipA-AuNP, when used inconjunction with the exemplary potent cancer chemotherapeutic drugdoxorubicin suppresses tumor growth.

“SipA” and “Salmonella T3SS effector protein” are used interchangeablyto refer to a protein produced by Salmonella, as exemplified by theamino acid sequence SEQ ID NO:01 of Salmonella enterica subsp. entericaserovar Typhimurium str. SL1344 (GenBank: AAA86618.1) (FIG. 7) encodedby the DNA sequence (Locus taq) SL1344_2861 of the Salmonella entericasubsp. enterica serovar Typhimurium str. SL1344, complete genomesequence (NCBI Reference Sequence: NC_16810.1).

Several biological activities have been identified for SipA. Forexample, SipA has been shown to participate in actin polymerization andbacterial invasion (Zhou D et al. Science. 1999 Mar. 26;283(5410):2092-5; Schumberger M C. Et al., Mol Microbiol. 2007 August;65(3):741-60). SipA has been shown to be involved in pro-inflammatoryresponses, such as neutrophil recruitment (Wall, D. et al. CellMicrobiol. 2007 September; 9(9):2299-313; Silva M., et al., Am J PhysiolGastrointest Liver Physiol. 2004 June; 286(6):G1024-31; Criss A K. etal., J Biol Chem. 2001 Dec. 21; 276(51):48431-9; and Lee C A. et al.,Proc Natl Acad Sci USA. 2000 Oct. 24; 97(22): 12283-8), activation ofthe NOD1/NOD2 signaling pathway (Keestra A M, et al., MBio. 2011 Dec.20; 2(6)), and CXC chemokine expression (through p38MAPK and JUNpathways (Figueiredo J F. et al. Microbes Infect. 2009 February;11(2):302-10), SipA has also been shown to be active in Mrp2up-regulation and HXA3 axis (Pazos M, et al., J Immunol. 2008 Dec. 1;181(11):8044-52; Agbor, T., et al., Cell Microbiol. 2011 December;13(12):2007-21; and Mrsny R J. et al., Proc Natl Acad Sci USA, 2004 May11; 101(19):7421-6).

The active sites in SipA that are associated with its several biologicalfunctions have also been mapped. For example, the active sites for SipAactin polymerization and bacterial invasion activity are located in thecarboxyl-terminal (ABD domain amino acid 446-685 of SEQ ID NO:01)(Galkin, V. et al., Nature Structural Biology 9, 518-521 (2002)). Theactive sites for SipA actin polymerization and bacterial invasion arealso located in the central region of SipA (amino acid 105-446 of SEQ IDNO:01), including the F1 (amino acid 170-271 of SEQ ID NO:01) which isrequired for initiation of SipA focus formation and cooperates with theABD domain, and the F2 (amino acid 280-394 of SEQ ID NO:01) whichenhances focal accumulation of SipA presumably via intermolecularSipA-SipA interactions (Schumherger M C. Et al., Mol Microbiol, 2007August; 65(3):741-60). The active sites for SipA actin polymerizationand bacterial invasion are also located in the N-terminal SipA region(amino acid 1-105 of SEQ ID NO:05) which mediates TTSS-1 transport(Bronstein, P. et al., Bacteriol. 2000 December; 182(23): 6638-6644).

The active site for SipA neutrophil recruitment activity are located inSipAa3 (amino acid 294-424 of SEQ ID NO:01) which is a 131-amino-acidregion (Wall, D. et al., Cell Microbiol. 2007 September; 9(9):2299-313).

3. Methods for Reduces Disease Symptoms

In one embodiment, the invention provides a method for reducing one ormore symptoms of a disease in a mammalian subject in need thereof(including at risk for disease), wherein the disease is associated withcells that overexpress one or more of P-gp and p53, the methodcomprising administering to the subject a composition comprising SipA,and/or polypeptide sequence having at least 95% identity to SipA (SEQ IDNO:01), wherein SipA (and/or the polypeptide sequence) is in an amountthat is effective in one or more of a) reducing the level of expressionof P-gp in the cells, b) reducing the level of functional un-cleavedP-gp in the cells, and c) increasing the level of expression of PERP inthe cells. In one embodiment, SipA and/or the polypeptide sequencehaving at least 95% identity to SipA is purified.

Thus in one embodiment, data in Examples 2 and 3 demonstrated that SipAis effective in reducing the level of expression of P-gp in the cells.

In another embodiment, Example 4 shows that SipA reduces the level offunctional un-cleaved P-gp by increasing the level of cleavage of P-gpby caspase-3 (CASP3), thus increasing the level of P-gp cleavageproducts that comprise the approximately 90 kDa P-gp cleavage productand/or the approximately 60 kDa P-gp cleavage product.

In a further embodiment, SipA increases the level of expression of PERPin cells, PERP is a tetraspan membrane protein originally identified asa transcriptional target of the p53 tumor suppressor (10). P53 regulatesthe cell cycle and, thus, functions as a tumor suppressor that isinvolved in preventing cancer. As such, p53 has been described as “theguardian of the genome”, referring to its role in conserving stabilityby preventing genome mutation. Fundamental to the tumor-suppressor roleof p53 is the ability to engage in apoptosis. This notion is stronglysupported by studies revealing the presence of p53 mutations in overhalf of human cancers (11, 12), and the compromised p53 activity (byother mechanisms) in the majority of other cancers (12). Studiesinvestigating the interaction of SipA with the surface epithelial cellshave been carried out (13, 14). The inventors used a split-ubiquitinbased yeast-two hybrid analysis system (Dualsystems Biotech) with fulllength SipA as bait and a human cancer colon mRNA-based library as prey,to identify PERP, a p53 induced apoptotic effector, as a SipAinteracting partner. Not only does SipA bind to PERP at epithelialsurfaces, but it also up-regulates the protein expression of PERP inhuman colonic cancers in vitro. While an understanding of the mechanismis not necessary, and without limiting the invention to any particularmechanism, and although the precise function of PERP in eliciting anapoptotic response remains unknown, initial reports indicate that PERPexpression causes nuclear localization of p53 and increases the level oftranscriptionally active p53 protein. In addition, other studies havefound that increased PERP expression affects several aspects of p53regulation. Including increased protein stability, postranslationalmodifications, and enhanced nuclear accumulation. These observationsplace PERP at a critical signaling circuit by influencing pathways ofp53 activation, and underscore a unique role for this protein inenhancing functional p53 levels and in increasing p53 stability. A lossof PERP expression promotes tumorigenesis (15). Since SipA promotes theproduction of PERP the inventors can exploit this natural response as ameans to enhance p53 (apoptotic) activity. p53-based drugs have beenshown to modify a variety of survival metrics resulting in inhibition ofcell proliferation, selective apoptosis in tumor cells, and completetumor growth inhibition.

In one embodiment, the diseases that are amenable to therapy using anyone of the inventions methods include, without limitations, cancer,neuroinflammation (such as multiple sclerosis) (Kooij, G et al., PLoSOne. 2009; 4(12): e8212), autoimmune disease (Van de Ven, R. et al., JLeukoc Biol. 2009 November; 86(5):1075-87), and infection with HumanImmunodeficiency Virus (HIV) (Jones, K. et al., AIDS. 2001 Jul. 27;15(11): 1353-8).

In a particular embodiment, the invention provides a method for reducingone or more symptoms of cancer in a mammalian subject in need thereof,comprising administering to the subject a composition comprising SipA,and/or polypeptide sequence having at least 95% Identity to SipA (SEQ IDNO:01). Data herein in Example 6 show that SipA conjugated to goldnanoparticles (SipA-AuNP) improved doxorubicin efficacy in the exemplarymurine colon cancer animal model.

In one embodiment, SipA and/or the polypeptide sequence having at least95% identity to SipA is purified.

The invention's methods are advantageously applicable to cancers thatcontain cancer cells resistant to at least one cytotoxin.

In one embodiment, the cancer comprises cancer cells that overexpressP-gp and/or p53 compared to a control cell.

In one preferred embodiment, the cancer cells overexpress P-gp. This isexemplified by colorectal cancer (Hota, T. et al,Hepatogastroenterology. 1999 January-February; 46(25):316-21), breastcancer (Bruce, J, et al., JNCI J Natl Cancer Inst (1997) 89 (13):917-93), bladder cancer (Tada, Y. et al., Int J Cancer. 2002 Apr. 1;98(4):630-5), sarcomas (including osteosarcoma) (Chan, H S. Et al., JNatl Cancer Inst. 1997 Nov. 19; 89(22):1706-15), astrocytoma (bloodbrain barrier) (Sadanand et al., Cancer Lett. 2003 Jul. 30;198(1):21-7), hematological malignancies such as acute myelogenousleukemia (AML) (Leith, C P. et al. Blood. 1999 Aug. 1; 94(3):1086-99)and diffuse large B-cell lymphoma (Yagi, K. et al., Histopathology; 2013February; 62(3):414-20).

In another preferred embodiment, the cancer cells contain a TP53mutations. TP53 is the most frequently altered gene in human cancers, itis inactivated in about 50% of human cancers (T. Soussi, C. Béroud,Assessing TP53 status in human tumours to evaluate clinical outcome,Nat. Rev. Cancer, 1 (2001), pp. 233-240).

The invention's compositions are preferably administered in atherapeutic amount. The terms “therapeutic amount,” “pharmaceuticallyeffective amount,” “therapeutically effective amount,” “biologicallyeffective amount,” and “protective amount” are used interchangeablyherein to refer to an amount that is sufficient to achieve a desiredresult, whether quantitative auditor qualitative. In particular, atherapeutic amount is that amount that delays, reduces, palliates,ameliorates, stabilizes, prevents and/or reverses one or more symptomsof the disease compared to in the absence of the composition ofinterest. Examples include, without limitation, tumor size and/or tumornumber in cancer disease.

For example, specific “dosages” of a “therapeutic amount” will depend onthe route of administration, the type of subject being treated, and thephysical characteristics of the specific subject under consideration.These factors and their relationship to determining this amount are wellknown to skilled practitioners in the medical, veterinary, and otherrelated arts. This amount and the method of administration can betailored to achieve optimal efficacy but will depend on such factors asweight, diet, concurrent medication and other factors, which thoseskilled in the art will recognize. The dosage amount and frequency areselected to create an effective level of the compound withoutsubstantially harmful effects.

The dosage is adjusted depending on the type and severity of thedisease, and, for example, whether there are one or more separateadministrations, or continuous infusion. For repeated administrationsover several days or longer, depending on the condition, the treatmentis repeated until a desired suppression of disease symptoms occurs.

The invention's compositions may be administered prophylactically (i.e.,before the observation of disease symptoms) and/or therapeutically(i.e., after the observation of disease symptoms). The term“administering” to a subject means providing a molecule to a subject.This may be done using methods known in the art (e.g., Erickson et al.,U.S. Pat. No. 6,632,979; Furuta et al., U.S. Pat. No. 6,905,839;Jackobsen et al., U.S. Pat. No. 6,238,878; Simon et al., U.S. Pat. No.5,851,789). Administration may be concomitant with (i.e., at the sametime as, or during) manifestation of one or more disease symptoms. Also,the invention's compositions may be administered before, concomitantlywith, and/or after administration of another type of drug or therapeuticprocedure (e.g., surgery). Methods of administering the invention'scompositions include, without limitation, administration in parenteral,oral, intraperitoneal, intranasal, topical and sublingual forms.Parenteral routes of administration include, for example, subcutaneous,intravenous, intramuscular, intrastemal injection, and infusion routes.In a particular embodiment, administration is intraperitoneal (seeExample 6).

In some embodiments, the invention's compositions may comprise lipidsfor delivery as liposomes. Methods for generating such compositions areknown in the art (Borghouts et al. (2005). J Pept Sci 11, 713-726; Changet al. (2009) PLoS One 4, e4171; Faisal et al. (2009) Vaccine 27,6537-6545; Huwyleret al. (2008) Int J Nanomedicine 3, 21-29; Song et al.(2008) Int J Pharm 363, 155-161; Voinea et al. J Cell Mol Med 6,465-474), US 2011/0129526 A1.

In one embodiment, the invention's compositions may comprisenanoparticles, microspheres, microparticles, and microcapsules fordelivery, using methods known in the art (US 2011/0129526 A1). In onpreferred embodiment, the invention's compositions may comprisenanoparticles.

The invention's methods may further comprise administering to thesubject one or more cytotoxin. Data herein in Example 6 show asurprising synergistic effect between the cytotoxin doxorubicin andSipA-AuNP, since P-gp expression levels in tumors that received only theSipA-AuNP treatment were modestly reduced (about 10%), wherein thecombination of cytotoxin doxorubicin and SipA-AuNP resulted in asignificant reduction in p-gp expression levels in tumors (about 40%).

The cytotoxin may be administered before and/or concomitantly withand/or after administration of SipA. Example 6 shows that SipAconjugated to gold nanoparticles improved doxorubicin efficacy in amurine colon cancer animal model.

In some embodiments, SipA is administered in a therapeutic amount thatis effective in one or more of a) reducing the level of expression ofP-gp in cells of the cancer, b) reducing the level of functionalun-cleaved P-gp in cells of the cancer, and c) increasing the level ofexpression of PERP in cells of the cancer.

For example, SipA may be administered in a therapeutic amount that iseffective in reducing the level of expression of P-gp in cells of thecancer (see Examples 2 and 3).

Also, SipA may be administered in a therapeutic amount that is effectivein reducing the level of functional un-cleaved P-gp in cells of thecancer. Thus, Example 4 shows that SipA reduces the level of functionalP-gp by increasing the level of cleavage of P-gp by caspase-3 (CASP3),thus increasing the level of P-gp cleavage products that comprise theapproximately 90 kDa P-gp cleavage product and/or the approximately 60kDa P-gp cleavage product.

In some embodiments, reducing the level of expression of P-gp comprisesa reduction of from 10% to 100% in the mammalian cell compared to in theabsence of administering SipA. A reduction of from 10% to 100% includes,for example, a reduction of 10%, 15%, 20% s 25%, 30% s 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%. Data hereinin Example 3 demonstrate a reduction of P-gp expression by 40% and 95%in breast cancer cells and bladder cancer cells, respectively, followingadministration of purified SipA.

In some embodiments, the methods further comprise determining the levelof expression of P-gp in the mammalian cell, using exemplary methodsdescribed in Examples 2 and 3. In some embodiments, the method furthercomprises determining the level of functional un-cleaved P-gp in themammalian cell. This may include, for example, determining the level ofone or more portions of P-gp, including a P-gp fragment that is producedby cleavage of P-gp with caspase-3. In some embodiments, the P-gpfragment comprises one or more of an approximately 90 kDa P-gp cleavageproduct and an approximately 60 kDa P-gp cleavage product. Data hereinin Example 4 show that SipA reduces the level of functional P-gp byincreasing the level of cleavage of P-gp by caspase-3 (CASP3), thusincreasing the level of P-gp cleavage products that comprise theapproximately 90 kDa P-gp cleavage product and/or the approximately 60kDa P-gp cleavage product.

In another embodiment, the methods further comprises determining thelevel of expression of PERP in the mammalian cell.

In particularly preferred embodiments, SipA is operably conjugated to acytotoxin (e.g., doxorubicin.

In a further embodiment, SipA is operably conjugated to a targetingagent that specifically binds to the cell. As used herein, the term“targeting agent” refers to a chemical moiety that, when associated with(i.e., covalently coupled or otherwise stably associated with) anothermoiety (such as a therapeutic molecule) in a complex, directs thecomplex to a specific site where the complex can then be imaged and/orwhere the complex delivers its associated therapeutic molecule. Suitabletargeting agents are known in the art. Representative targeting agentsare one of a binding pair. For example, in one embodiment, the targetingagent is an antibody, an antigen-binding portion of the antibody, or itsantigen. The antigen can be a small molecule, peptide, protein,polynucleotide, or polysaccharide. In one embodiment, the targetingagent is a nucleic acid or its complement. The nucleic acids can be DNAsand RNAs. In one embodiment, the targeting agent is an enzyme or itssubstrate. In one embodiment, the targeting agent is a receptor or itsligand. In one embodiment, the targeting agent is a nucleic acid or itspartner protein. In one embodiment, the targeting agent is a ligand fora cell, a cell membrane, or an organelle.

In one embodiment, targeting agents that specifically binds to a cancercell include folic acid, and “cyclic-arginine-glycine-aspartic acid”(“cRGD”) peptide. RGD-4C-Peptide has been shown to specifically bind tohuman breast cancer cells as well as to cancer endothelial cells, invivo (Zitsmann et al. Cancer Res Sep. 15, 2002 62; 5139).

Antibodies that specifically bind to cancer cells are known in the artincluding those specific for breast cancer (US 2004/0151724),prostate-specific membrane antigen (PSMA) antibody specific for prostatecancer (WO 2011/057146), EGFR antibody specific for glioblastoma (WO2011/057146), AFAI antibody specific for lung cancer (US 2009/0226942and US 2006/0159687 and WO 2004/078097), urinary tumor associatedantigen (UTAA) specific antibodies such as TA90 specific antibodies(U.S. Pat. Nos. 5,700,649 and 5,993,828, US 2010/0247440).

In certain embodiments, such as imaging or treating tumors, antibodiesof use may target tumor-associated antigens. These antigenic markers maybe substances produced by a tumor or may be substances which accumulateat a tumor site, on tumor cell surfaces or within tumor cells. Amongsuch tumor-associated markers are those disclosed by Herberman,“Immunodiagnosis of Cancer”, in Fleisher ed., “The Clinical Biochemistryof Cancer”, page 347 (American Association of Clinical Chemists, 1979)and in U.S. Pat. Nos. 4,150,149; 4,361,544; and 4,444,744. Reports ontumor associated antigens (TAAs) include Mizukami et al., (2005, NatureMed, 11:992-97); Hatfield et al., (2005, Curr. Cancer Drug Targets5:229-48); Vallbohmer et al. (2005, J. Clin. Oncol. 23:3536-44); and Renet al. (2005, Ann. Surg. 242:55-63).

Another marker of interest is transmembrane activator andCAML-interactor (TACI). See Yu et al. Nat. Immunol. 1:252-256 (2000).

Where the disease involves a lymphoma, leukemia or autoimmune disorder,targeted antigens may be selected from the group consisting of CD4, CD5,CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38,CD40, CD40L, CD46, CD52, CD54, CD67, CD74, CD79a, CD80, CD126, CD138,CD154, B7, MUC1, Ia, Ii, HM1.24, HLA-DR, tenascin, VEGF, PIGF, ED-Bfibronectin, an oncogene (e.g., c-met or PLAGL2), an oncogene product,CD66a-d, necrosis antigens, IL-2, T101, TAG, IL-6, MIF, TRAIL-R1 (DR4)and TRAIL-R2 (DR5).

In a particularly preferred embodiment, the targeting agent isexemplified by an antibody, an antigen-binding portion of an antibody,cyclic-arginine-glycine-aspartic acid (cRGD) peptide, and folic acid.

The invention further provides a method comprising administering to amammalian cell a composition comprising SipA, and/or polypeptidesequence having at least 95% identity to SipA (SEQ ID NO:01), whereinSipA (and/or the polypeptide sequence) is in an amount that is effectivein one or more of a) reducing the level of expression of P-gp in thecell (see Examples 2 and 3), b) reducing the level of functionalun-cleaved P-gp in the cell (see Example 4, which shows that SipAreduces the level of functional P-gp by increasing the level of cleavageof P-gp by caspase-3 (CASP3), thus increasing the level of P-gp cleavageproducts that comprise the approximately 90 kDa P-gp cleavage productand/or the approximately 60 kDa P-gp cleavage product), and c)increasing the level of expression of PERP in the cell.

In one embodiment, SipA and/or the polypeptide sequence having at least95% identity to SipA is purified.

In some embodiments of the inventions' methods, the cell overexpressesone or more of P-gp and p53 compared to a control cell.

In particularly preferred embodiments, the cell that overexpresses p53is a cancer cell

In another particularly preferred embodiment, the cell thatoverexpresses P-gp is a cancer cell and/or a non-cancerous cell.

In some embodiments, the cell is in vitro and/or in vivo.

Exemplary non-cancerous cells that overexpress P-gp comprise alymphocyte cell, such as a lymphocyte infected with HIV.

In another embodiment, non-cancerous cells that overexpress P-gpcomprise an intestinal epithelial cell. Data herein in Example 6demonstrate that SipA significantly deceased expression of P-gp in micethat were infected with a Salmonella typhimurium strain thatoverexpressed SipA, compared to an isogenic S. typhimurium mutantstrain.

In some embodiments, the cancer cell that overexpresses P-gp comprisesone or more of a colon cancer cell (see Example 2), colorectal cancercell, gastro-intestinal cancer cell, breast cancer cell (see Example 3),bladder cancer cell (see Example 3), kidney cancer cell, leukemia cell,brain cancer cell, sarcoma cell, astrocytoma cell, acute myelogenousleukemia (AML) cell, and diffuse large B-cell lymphoma cell.

In some embodiments, the cancer cell is in vivo, and SipA administrationmay be oral, transdermal, intravenous, intraperitoneal (see Example 6),and/or by local injection.

In alternative embodiments, the cancer cell is in vivo, and SipAadministration is before and/or concomitantly with and/or afteradministration of a cytotoxin.

4. SipA Conjugated to Nanoparticles

The invention further provides a nanoparticle comprising one or morepurified SipA and/or one or more polypeptide sequence having at least95% identity to SipA (SEQ ID NO:01).

The functional design of the invention's nanocarrier particles wasfounded on the inventors' discovery that the Salmonella effectorprotein, SipA targets two pathways critical for improvingchemotherapeutic efficacy: multidrug resistance and stabilizing p53, atumor suppressor protein. The innovation of this technology and theunconventional nature of the approach was centered on the development ofa novel AuNP (gold nanoparticle) scaffold in which SipA was engineeredas part of a drug nanocarrier that works in combination with a knownchemotherapeutic drug, such as doxorubicin. In essence the inventorscreated a nanoparticle that acts as a bacterial mimic to reduce(including reverse) multidrug resistance.

The invention's methods that employ SipA conjugated to nanoparticles(such as the SipA-conjugated AuNP bacterial mimic) capitalize on theunique chemical and physical properties of Au-NPs, the biochemicalfunctional activities of SipA, and can be used as a stand-alonetreatment, and/or in conjunction with many different FDA approvedchemotherapeutic agent, such as doxorubicin. If desired, the SipA-AuNPsystem can additionally be conjugated with an acceptor molecule thatrecognizes its cognate receptor on the surface of target tumor cells;e.g., antibodies, cyclic-arginine-glycine-asparlic acid (cRGD) peptideand folic acid. Such conjugation further improves the tumor targetingcapabilities of the SipA-AuNP system to minimize unwanted off-targetp-gp effects. In sum, such bacterial mimics present a facile andpowerful system to overcome multidrug resistance in tumor chemotherapythat can be combined with different anti-cancer drugs to target avariety of cancers, including colon, breast, and leukemia.

The invention's compositions are useful in any one of the invention'smethods. In a particular embodiment, SipA is operably conjugated to ananoparticle. In another embodiment the nanoparticle is further operablyconjugated to a cytotoxin and/or targeting agent that specifically bindsto a cell. In some embodiments, the targeting agent that specificallybinds to the cell comprises one or more of antibody, antigen-bindingportion of an antibody, cyclic-arginine-glycine-aspartic acid peptide(cRGD).

In some embodiments the nanoparticle comprises a gold nanoparticle, suchas a nanoparticle from 1 nm to 100 nm. In a particularly preferredembodiment, the nanoparticle is 15 nm. Data herein in Example 5 describethe construction and use of exemplary 15 nm gold nanoparticlesconjugated to SipA via tetra ethylene glycol (TEG) surface ligandspacers.

In some embodiments, SipA is operably conjugated at a nanoparticle:SipAratio of at least 1:1. In one embodiment, the nanoparticle:SipA ratio isany ratio from 1:1 to 1:100 including from 1:5, from 1:2, from 1:3, from1:4, from 1:5, from 1:6, from 1:7, from 1:8, from 1:9, from 1:10, from1:11, from 1:12, from 1:13, from 1:14, from 1:15, from 1:16, from 1:17,from 1:18, from 1:19, from 1:20, etc. In a preferred embodiment, thenanoparticle:SipA ratio is 1:6. Data herein in Example 5 demonstrate thesuccessful construction of SipA conjugated to gold particles at ananoparticle:SipA ratio of 1:6, and the successful use of theseparticles to reduce the level of P-gp expression in cancer cells at SipAdoses that are nearly 500 times lower than in free unbound SipA (FIG.5B).

In some embodiments, the invention's compositions comprise any one ormore of the nanoparticles described herein and at least onepharmaceutically acceptable molecule, such as diluent and/or excipient.

Exemplary “diluent” (“carrier”) includes water, saline solution, humanserum albumin, oils, polyethylene glycols, aqueous dextrose, glycerin,propylene glycol or other synthetic solvents. Diluents may be liquid(such as water, saline, culture medium, saline, aqueous dextrose, andglycols) or solid carriers (such as carbohydrates exemplified by starch,glucose, lactose, sucrose, and dextrans, anti-oxidants exemplified byascorbic acid and glutathione, and hydrolyzed proteins).

An “excipient” is an inactive substance used as a carrier for theinvention's compositions that may be useful for delivery, absorption,bulking up to allow for convenient and accurate dosage of theinvention's compositions. Excipients include, without limitation,antiadherents, binders (e.g., starches, sugars, cellulose, modifiedcellulose such as hydroxyethyl cellulose, hydroxypropyl cellulose andmethyl cellulose, lactose, sugar alcohols such as xylitol, sorbitol andmaltitol, gelatin, polyvinyl pyrrolidone, polyethylene glycol), coatings(e.g., shellac, corn protein zein, polysaccharides), disintegrants(e.g., starch, cellulose, crosslinked polyvinyl pyrrolidone, sodiumstarch glycolate, sodium carboxymethyl cellulose), fillers (e.g.,cellulose, gelatin, calcium phosphate, vegetable fats and oils, andsugars, such as lactose), diluents, flavors, colors, glidants (e.g.,silicon dioxide, talc), lubricants (e.g., talc, silica, fats, stearin,magnesium strearate, stearic acid), preservatives (e.g., antioxidantssuch as vitamins A, E, C, selenium, cystein, methionine, citric acids,sodium citrate, methyl paraben, propyl paraben), sorbents, sweeteners(e.g., syrup). In one embodiment, the excipient comprises HEC(hydroxyethylcellulose), which is a nonionic, water-soluble polymer thatcan thicken, suspend, bind, emulsify, form films, stabilize, disperse,retain water, and provide protective colloid action. HEC isnon-inflammatory and has been used as a delivery vehicle for vaginalmicrobiocides (Tien et al., AIDS Research & Human Retroviruses, (2005),21:845).

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

Example 1 Materials and Methods

The following is a brief description of the exemplary materials andmethods used in the subsequent Examples.

Chemicals. Anti-P-gp mouse mAb C219 and C494 were purchased fromCalBiochem (La Jolla, Calif.). The CASP3 inhibitor (SC-3075) and CASP1inhibitor (SC-3071) were purchased from Santa Cruz Biotechnology (SantaCruz, Calif.). The Anti-HA affinity matrix and HA peptide were purchasedfrom Roche applied science (Mannheio, Germany).

Cell culture. The human intestinal adenocarcinoma cell line HCT8 weremaintained in accordance with (8). The human breast adenocarcinoma cellline MCF-7, the human bladder transitional cell carcinoma cell lineUM-UC-3, and the CT26 murine colon carcinoma cell line, were purchasedfrom ATCC and were all maintained in DMEM F-12 containing a 10% fetalbovine serum, 100 U/ml penicillin, and 10 μg/ml streptomycin at 37° C.in 90% relative humidity and 5% CO₂.

Bacterial strains, plasmids and growth conditions. All S. Typhimuriumstrains are derived from SL1344. The AKJ63 strain has been previouslydescribed (22). Additionally, ΔSipC, ΔSipB, and ΔSopA have all beenpreviously described (9, 11).

Isolation of S. Typhimurium secreted proteins. Wild type S. TyphimuriumSL1344 or mutants were grown in LB medium overnight in accordance with(22). The proteins from the culture supernatants were precipitated with10% (vol/vol) trichloroacetic acid, as previously described (9).

The purification of SipA-HA fusions protein. The purification of SipAwas performed in accordance with the work of Lee et al, (22).

Cell lysates and Western Blot analysis. Cell lysates were harvested fromS. Typhimurium-infected HCT8 cells, as previously described (8).Proteins were normalized to 30 μg, separated by SDS/PAGE (4-12%gradient; Biorad, Hercules, Calif.), and transferred to nitrocellulose(Bio-Rad; 0.45μ membrane). Immuno-blots were performed using the murinemonoclonal P-gp C219 antibody (calbiochem) diluted at 1:100. A goatanti-mouse IgG labeled with horseradish peroxidase (Santa Cruz, Calif.)diluted at 1:10000 was used to detect the bands, which were visualizedby enhanced chemiluminescence using a super signal West pico kit(Thermo, Rockford, Ill.).

SipA-AuNP conjugation chemistry:

1. A synthesis of the dithiolated tetra (ethylene glycol) carboxylicacid.

A synthesis of Undec-1-en-11-yltetra (ethylene glycol). A mixture of0.34 ml of 50% aqueous sodium hydroxide (4.3 mmol) and 4.08 g of tetra(ethylene glycol) (21 mmol) was stirred for about 0.5 h in an oil bathat 100° C. under an atmosphere of argon, and then 1.0 g of11-bromoundec-1-ene (4.3 mmol) was added. After 24 h, the reactionmixture was cooled and extracted six times with hexane. Concentration ofthe combined hexane portions by rotary evaporation at reduced pressuregave yellow oil containing a mixture of mono- and diethers, according toanalysis by ¹HNMR spectroscopy. Purification of the oil bychromatography on silica gel (eluant: ethyl acetate) gave 0.98 g ofmonoether: 76% yield; ¹H NMR (400 MHz, CDCl) 1.22-1.27 (m, 10H),1.29-1.34 (m, 2H), 1.49-1.56 (m, 2H), 1.96-2.02 (m, 2H), 2.73-2.76 (t,1H), 3.38-3.42 (t, 2H, J=7 Hz), 3.52-3.69 (m, 16H), 4.86-4.97 (m, 2H),5.71-5.82 (m, 1H). MS (ESI-MS) calcd for C₁₉H₃₈O₅ 346.50, found 347.2[M+H]⁺.

To a solution of Undec-1-en-11-yltetra(ethylene glycol) (1.0 g 2.89mmol) in dry DCM (6 mL) at 0° C. was added ethyl diaxoacetate (0.7 mL,5.78 mmol) and BF₃Et₂O (0.29 mmol). After the mixture was stirred for 30min at 0° C. saturated ammonium chloride (3 mL) was added and thereaction mixture was placed in a separated funnel. The organic phase wascollected and the aqueous phase was extracted with DCM (5*150 mL). Thecombined organic phase was dried over Na₂SO₄ and concentrated to ayellow oil, which was purified by chromatography using gradient elutionhexane (1:1) to ethyl acetate to offered ester. ¹H NMR (400 MHz, CDCl)1.19-1.22 (m, 13H), 1.26-1.31 (m, 2H), 1.46-1.52 (m, 2H), 1.93-1.98 (m,2H), 3.35-3.38 (t, 2H, J=7 Hz), 3.52-3.69 (m, 16H), 4.11-4.16 (m, 2H),4.07 (s, 2H), 4.82-4.93 (m, 2H), 5.69-5.77 (m, 1H). MS (ESI-MS) calcdfor C₂₃H₄₄O₇432.31, found 450.2 [M+H₃O]⁺.

To a solution of ester (0.10 g, 0.23 mmol) in dry DCM (10 mL) was addedbromine (0.28 mmol) at 0° C. The reaction mixture was stirred at 0° C.for 4 hours at the dark. Thereafter, the reaction mixture was isolatedby removal of the solvent using a slight vacuum and a water bathtemperature of 30° C. in a rotary evaporator and final drying of theproduct in vacuum. ¹H NMR (400 MHz, CDCl) 1.22-1.38 (m, 13H), 1.49-1.61(m, 4H), 1.71-1.80 (m, 2H), 3.42-3.47 (t, 2H, J=7 Hz), 3.58-3.77 (m,17H), 3.81-4.87 (m, 1H), 4.17 (s, 2H), 4.19-4.22 (m, 3H). MS (ESI-MS)calcd for C₂₃H₄₄Br₂O₇ 592.40, found 615.2 [M+Na]⁺.

A solution of dibromine (100 mg, 0.17 mmol) and K₂CO₃ (117 mg, 0.85mmol) in acetone (10 mL) was added thioacetic acid (129 mg, 1.7 mmol).The reaction mixture was stirred at room temperature overnight, ¹H NMR(400 MHz, CDCl,) 1.09-1.35 (m, 13H), 1.48-1.68 (m, 4H), 1.93-2.01 (m,2H), 2.32 (s, 6H), 3.08-3.28 (m, 1H), 3.39-3.50 (m, 3H), 3.55-3.78 (m,17H), 4.15 (s, 2H), 4.18-4.24 (m, 2H), MS (ESI-MS) calcd forC₃₇H₅₀O₉S₂582.81, found 621.3 [M+K]⁺.

The solution of diactyl-OEt in ethyl was then added concentratedhydrochloric acid and stirred overnight to provide free thiol compound.¹H NMR (400 MHz, CDCl,) 1.18-1.38 (m, 10H), 1.49-1.61 (m, 8H), 2.72-2.98(m, 3H), 3.36-3.42 (t, 2H, J=7 Hz), 3.55-3.77 (m, 16H), 4.17 (s, 2H). MS(ESI-MS) calcd for C₂₁H₄₂O₇S₂ 470.68, found 471.3 [M+H]⁺.

2. Syntheses of the Au—COOH.

15 nm of AuNPs were first synthesized using citrate as a reducing agentand stabilizer. HAuCl4 (10 mg) was dissolved in 90 ml of water, and thesolution was heated to the boiling point. Sodium citrate solution (500μl of 250 mM) was added to the boiling solution and stirred for 30minutes until the color turned to wine-red. The resulting AuNP was thenwashed three times. Five mg of the dithiolated tetra (ethylene glycol)carboxylic acid was subsequently mixed with 10 pmoles of AuNPs in 5 mlof water, leading to an overnight ligand change reaction. The affordedAu—COOH nanoparticles were dialyzed in DI water using a Slide-A-LysserMINI dialysis unit (MW=10,000) for two days.

3. Conjugation and characterization of the SipA conjugated AuNPs.

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) 10 mg andN-hydroxysuccinimide (NHS) 10 mg were added to a 28 ml (7.20 pmols)solution of Au—COOH. The resultant solution was stirred at roomtemperature. After 1 h, the solution was centrifuged and washed threetimes with DI water. Finally, the solution was concentrated to 2 ml ofDI water, 200 μl (540.7 μg/ml) SipA solution was added to this solution.This mixed solution was stirred in a cold room that was 4° C. After 12h, the solution was centrifuged and washed three times with DI water.Finally, the SipA-AuNP stock solution was concentrated to 0.5 ml of DIwater and was then dialyzed with Slide-A-Lyzer MINI dialysis unit(MW=100,000) in DI water overnight.

The subcutaneous tumor model. Female 8 to 10 week old Balb/C mice werepurchased from Jackson Laboratory (Bar Harbor, Me.) and allowed toacclimatize for 4 days. CT26 cells were harvested by trypsin treatment,washed twice in PBS buffer, and resuspended in PBS. CT26 cells (5×10⁵)were inoculated in 100 μl subcutaneously in to the right flank (16).Mice were randomly assigned to the control group (n=6) or the treatmentgroups (n=6)/group. After several days, the mice harbored tumors withvolumes of ˜0.5 mm³, and were IP injected with 1.1 μg/day of SipA-AuNP(200 ul) for two days. The following day, the mice received a one-timedrug treatment of Doxorubicin (10 mg/kg) delivered by IP injection,followed by 1.1 μg/day of SipA-AuNP (IP) every 48 hours for 15 days. Twoweeks post drug delivery the mice were sacrificed and the tumors wereextracted for analysis, in regard to tumor size and expression of P-gp.Tumor size was measured using calipers and volumes were estimated usingthe formula 0.5×length×(width)². The care of these animals was inaccordance with University of Massachusetts Medical School Institutionalguidelines under protocol number: 2046-12. Statistical analysis wasperformed using Prism software (GraphPad).

Mouse infections. Mouse Infections were performed as previouslydescribed (9).

Example 2 SipA Modulates the Expression of P-gp in Intestinal EpithelialCells

Since the inventors' prior work revealed that S. Typhimurium SPI-1 isnecessary to down regulate the expression of P-gp (8), the inventorsbegan by screening S. Typhimurium type III secreted effectors todetermine if any are altered in their ability to modulate P-gp. theinventors found that when HCT8 human intestinal carcinoma cellmonolayers were exposed to Salmonella mutants of the type III secretionsystem translocon, ΔSipB or ΔSipC, these mutants maintained the abilityto modulate P-gp (FIG. 1A). Phenotypically, these mutants are able tosecrete effectors but they fail to translocate them into the host cells(9). the inventors' findings therefore suggest that a secreted effectorcould be modulating P-gp as a result of an extracellular interactionrather than due to its direct delivery into epithelial cells. To examinethis possibility, HCT8 epithelial monolayers were exposed to an extractof secreted proteins that were isolated from S. Typhimurium (Example 1).Since the treatment of this protein extract alone is sufficient totrigger the modulation of P-gp (FIG. 1B), the inventors next examinedindividual S. Typhimurium type III secreted effector mutants toestablish whether any fail to modulate P-gp. As shown in FIG. 1C, a S.Typhimurium ΔSipA mutant strain (EE633) is dramatically reduced in itsability to modulate P-gp. The specificity of the ΔsipA mutant defect wasverified by demonstrating that a plasmid that expresses the sipA generestores the ability of the ΔsipA mutant to modulate P-gp to theapproximate levels elicited by the wild-type strain (FIG. 1C).

To examine whether SipA alone can induce the modulation of P-gp withoutthe assistance of other Salmonella or type III effectors, purifiedSipA-HA was added to buffer overlying washed HCT8 cells. Exposure ofcell monolayers to 80 μg/ml or 160 μg/ml of SipA-HA over a period of 3hours resulted in a dose dependent ability to modulate P-gp to the samedegree as wild-type S. Typhimurium, This effect was not attributed totrace amounts of lipopolysaccharide (FIGS. 2A and B). Moreover, tofurther validate that SipA was responsible for the modulation of P-gp,monolayers were exposed to an extract of secreted proteins isolated fromthe S. Typhimurium ΔSipA mutant (Example 1). This extract contained allS. Typhimurium secreted effectors with the exception of SipA, and asshown in FIG. 2C, felled to modulate P-gp. Monolayers were also exposedto a secreted protein extract from the regenerated mutant, S.Typhimurium ΔSipA/pSipA, which was rescued in its ability to modulateP-gp (FIG. 2C).

Example 3 SipA Modulates the Expression of P-gp in Breast and BladderHuman Cancer Cell Lines

Because P-gp expression is documented to be up-regulated in severaltypes of malignancies, and contributes to their poor prognosis (6, 7),the inventors assessed whether the ability of SipA to down-regulate P-gpis broad spectrum. Similar to colonic cancer cell lines, purifiedSipA-HA was exposed to cell monolayers of different cancer cell typesthat are also known to over-express P-gp, such as MCF-7 (breastadenocarcinoma), and UM-UC-3 (human bladder carcinoma). Compared to thebuffer control, the exposure of purified SipA to MCF-7 cells reduced theexpression of P-gp in a dose dependent manner demonstrating a 40%reduction. Likewise, the inventors also found that SipA-HA modulates theexpression of P-gp on UM-UC-3 cells, showing a 95% reduction. Exposureof SipA-HA to HCT8 cells served as the positive control (FIGS. 3A andB). These results confirm that the ability of SipA to modulate P-gp isnot restricted to intestinal epithelial cells.

Example 4 Mechanism of SipA Action on P-pg

Our prior studies revealed that protein expression of P-gp isdown-regulated in Salmonella-infected epithelial cells without acorresponding decrease in P-gp mRNA. This observation is consistent witha mechanism of P-gp protein cleavage and/or rearrangement from the cellmembrane rather than the regulation of gene expression. Recent studiesusing human T-lymphoblastoid CEM cells have shown that P-gp undergoescaspase-3-dependent cleavage during apoptosis (10), providing evidencethat cells are able to functionally down-regulate P-gp through amechanism involving protein cleavage/degradation. Since the inventorshave previously shown that the SipA effector protein is necessary andsufficient to promote the activation of caspase-3 (CASP3) (11), theinventors next examined the protein expression of P-gp in HCT8 cellsfollowing infection from S. Typhimurium in the absence and presence of apharmacological inhibitor of CASP3. As shown in FIG. 4A, Western blotanalysis demonstrates that caspase-3, but not CASP1 inhibition (whichwas used as the negative control), prevented S. Typhimurium fromdown-regulating P-gp. A similar outcome was also observed using HCT8cells knocked-down (small interfering RNA (siRNA;(11))) for theexpression of CASP3 (FIG. 4B), further supporting the contention thatP-gp undergoes CASP3-dependent cleavage as a means to functionallydown-regulate this transporter. Moreover, in silico modeling of mouseP-gp (which is 89% identical to the human P-gp) revealed twosurface-exposed CASP3 cleavage motifs (DQGD and DVHD; FIG. 4C). In linewith this observation, the inventors also found that HCT-8 cellsinfected with S. Typhimurium showed a progressive reduction in theexpression of P-gp, and this correlated with the appearance of thepredicted CASP3 cleavage products of P-gp (90 kDa, and approximately 60kDa), as calculated from the in silico model (FIGS. 4C and D). The lower25 kDa band was not resolved. Taken together, these observations suggestthat the ability of S. Typhimurium to modulate P-gp via SipA depends onits ability to activate CASP3, and is consistent with the inventors'previous findings showing that SipA activates CASP3.

Example 5 Construction of SipA-AuNP

Capitalizing on the ability of SipA to broadly down-regulate P-gp, theinventors next sought to engineer SipA conjugated nanoparticles as aneffective chemotherapeutic adjuvant, the inventors selected goldnanoparticles as a scaffold because these particles are inert/not toxic,(12, 13) easily synthesized and modified, and stabilize conjugatedpharmaceutics (e.g., proteins (14) and small molecule drugs (12)). Theinventors fabricated 15 nm gold nanoparticles for this work sincenanoparticles that are less than 100 nm have a unique enhancedpermeation and retention (EPR) effect, and can, therefore effectivelyextravasate and remain within interstitial spaces, resulting in a muchhigher concentration of SipA at tumor sites (15).

Although substantial progress has been made in promoting the use ofAuNPs for genetic material and as small molecular drug delivery systems,the delivery of functional proteins with retention or enhanced activityhas been challenging due to inadequate maintenance of proteinrecognition and structure retention. To overcome this limitation, theinventors designed surface ligands for direct conjugation of SipA to theAuNP by inserting biocompatible tetra (ethylene glycol) (TEG) spacers(Example 1 and FIG. 5A). This adaptation reduces non-specificinteractions and absorption, and provides additional degrees of freedomand polyvalency for enhancing the conjugated protein's activity.Moreover, the carboxylate terminus creates a platform for subsequentprotein coupling. Lastly, the inventors covalently attached the SipAproteins to the carboxyl modified AuNP in order to avert proteindissociation or aggregation (Example 1 and FIG. 5A).

To determine the ratio of AuNP to surface conjugated SipA proteins, theinventors next exposed the SipA-AuNP to sodium cyanide, which decomposesthe gold particle core. This mixture was then dialyzed for two daysusing a Slide-A-Lyzer MINI dialysis unit (MW=10,000), and thereafterconcentrated to 45 μl for mass spectrometry characterization. Based onmass spectrometry analysis, the total amount of SipA protein wasdetermined to be 42.2 pmols, establishing the binding ratio of AuNP:SipAat 1:6 (Example 7). Subsequent in vitro testing of the SipA-conjugatedAuNP revealed that the design of this novel nanoparticle profoundlyincreases the stability of surface bound SipA protein and reduces P-gpexpression in cancer cells at SipA doses that are nearly 500 times lowerthan in free unbound SipA (FIG. 5B). Such enhanced SipA functionality ismost likely due to the large surface (the volume ratio of AuNP), whichdramatically stabilizes SipA proteins by preventing the conjugatedproteins from degradation. Additionally, the polyvalency of SipAproteins on the surface of single AuNP may offer a synergisticcooperation effect, which does not exist in free-bound SipA.

Example 6 SipA-AuNP Improves the Efficacy of Doxorubicin in a MurineColon Cancer Model

We next sought to determine whether the SipA-AuNP conjugate improves theefficacy of doxorubicin, a known chemotherapeutic drug, the inventorsused a well-established subcutaneous murine colon cancer model as aprototypical model to study cancers that are known to overexpress P-gp(16) (17). Disease in this model is induced by the subcutaneousinjection of CT26 colon cancer cells in ˜8 week old Balb/C mice. Theformation of palpable tumors (approximately 0.5 mm³ in size) denotes day1 of the experiment. Mice were then IP injected with 1.1 μg/day ofSipA-AuNP for two days prior to IP treatment of a single dose ofdoxorubicin (10 mg/kg). Since the key objective is to assess whetherSipA-AuNP itself is able to improve doxorubicin efficacy, 10 mg/kgidentifies a concentration of the drug that the inventors determineddisplays a minimal effect on tumor size. This treatment was followed bySipA-AuNP IP injections every 48 hours for 15 days, after which,doxorubicin efficacy was assessed by the tumor volume in mm³.

As shown in FIG. 6A, the tumor volume following the SipA-AuNP“nanobug”-doxorubicin combination treatment was significantly less thanthe tumor volumes following either SipA-AuNP or doxorubicin treatmentperformed alone (P<0.0001).

We found high concentrations of AuNPs in tumors treated with SipA-AuNPsand doxorubicin combination therapy (FIG. 6B), preferentially located inthe center of the tumor. Whereas, tumors treated with a tumors eitherSipA-AuNP or AuNP alone showed a diffuse distribution of the particles.This is consistent with the inventors' observation that P-gp expressionis profoundly diminished in tumors that received the SipA-AuNP and DOXcombination regiment (FIG. 6C). It is worth noting that the expressionof P-gp in tumors that received only the SipA-AuNP treatment was reducedmodestly (˜10%), It is likely that the different microenvironmentsencountered by the stable dose SipA-AuNP accounts for these findings.For example, in the SipA-AuNP and DOX combination treatment group, thesynergistic effects of P-gp Inhibition coupled with the chemotherapeuticdrug were marked by profound decreases in both the tumor size and thenumber of cells, which enabled the SipA-AuNP to further penetrate thetumor and act on cells at an effective concentration. In contrast, thegroup receiving only the SipA-AuNP regiments, encountered tumors with ahigh cell proliferation rate, which effectively diluted out the effectof the SipA-AuNPs. Consequently, these tumors did not exhibit modulatedP-pg levels.

Consistent with this notion, SipA could modulate the expression of P-gpin healthy murine intestinal epithelium in vivo. Since normal healthyintestinal epithelium display baseline expression of P-gp, the inventorsevaluated the colonic expression of P-gp in mice colonized with a S.Typhimurium strain that over-expresses SipA (AJK63) compared to micecolonized with an equivalent amount of an isogenic SipA mutant strain(EE633). Under these conditions, the inventors observed a significantdecrease in the expression of P-gp in mice that were infected with theSipA over-expressing strain as compared to the SipA mutant strain, thelatter of which felled to modulate the expression of P-gp (FIG. 6D).Taken together, these data provide evidence that the SipA protein isresponsible for in vivo P-gp down-regulation, the inventors alsoestablish the initial proof of concept that the SipA-AuNPs bacterialmimic, when used in conjunction with the potent cancer chemotherapeuticdrug doxorubicin, accumulates in tumors and promotes tumorregression/inhibition with a concomitant decrease in P-gp expression.

Example 7 Nanoparticles Conjugated to SipA A. Synthesis of theDithiolated Tetra (Ethylene Glycol) Carboxylic Acid

The Scheme of synthesis of the dithiolated tetra (ethylene glycol)carboxylic acid is shown in FIG. 8.

Briefly, a mixture of 0.34 mL of 50% aqueous sodium hydroxide (4.3 mmol)and 4.08 g of tetra (ethylene glycol) (21 mmol) was stirred for about0.5 h in an oil bath at 100° C. under an atmosphere of argon, and then1.0 g of 11-bromoundec-1-ene (4.3 mmol) was added. After 24 h, thereaction mixture was cooled and extracted six times with hexane.Concentration of the combined hexane portions by rotary evaporation atreduced pressure gave yellow oil containing a mixture of mono- anddiethers, according to analysis by 1HNMR spectroscopy. Purification ofthe oil by chromatography on silica gel (eluant: ethyl acetate) gave0.98 g of monoether (compound 1): 76% yield; 1H NMR (400 MHz, CDCl,)1.22-1.27 (m, 10H), 1.29-1.34 (m, 2H), 1.49-1.56 (m, 2H), 1.96-2.02 (m,2H), 2.73-2.76 (t, 1H), 3.38-3.42 (t, 2H, J=7 Hz), 3.52-3.69 (m, 16H),4.86-4.97 (m, 2H), 5.71-5.82 (m, 1H). MS (ESI-MS) calcd for C19H38O5346.50, found 347.2 [M+H]+.

To a solution of Undec-1-en-11-yltetra(ethylene glycol) (compound 1)(1.0 g 2.89 mmol) in dry DCM (6 mL) at 0° C. was added ethyldiazoacetate (0.7 mL, 5.78 mmol) and BF3Et2O (0.29 mmol). After themixture was stirred for 30 min at 0° C., saturated ammonium chloride (3mL) was added and the reaction mixture was placed in a separated funnel.The organic phase was collected and the aqueous phase was extracted withDCM (5×150 mL). The combined organic phase was dried over Na2SO4 andconcentrated to a yellow oil which was purified by chromatography usinggradient elution hexane (1:1) to ethyl acetate to offered ester,(compound 2) 1H NMR (400 MHz, CDCl,) 1.19-1.22 (m, 13H), 1.26-1.31 (m,2H), 1.46-1.52 (m, 2H), 1.93-1.98 (m, 2H), 3.35-3.38 (t, 2H, J=7 Hz),3.52-3.69 (m, 16H), 4.11-4.16 (m, 2H), 4.07 (s, 2H), 4.82-4.93 (m, 2H),5.69-5.77 (m, 1H), MS (ESI-MS) calcd for C23H44O7 432.31, found 450.2[M+H30]+.

To a solution of ester (compound 2) (0.10 g, 0.23 mmol) in dry DCM (10mL) was added bromine (0.28 mmol) at 0° C. The reaction mixture wasstirred at 0° C. for 4 hours at the dark. Thereafter, the reactionmixture was isolated by removal of the solvent using a slight vacuum anda water bath temperature of 30° C. in a rotary evaporator and finaldrying of the product in vacuum, (compound 3) 1H NMR (400 MHz, CDCl,)1.22-1.38 (m, 13H), 1.49-1.61 (m, 4H), 1.71-1.80 (m, 2H), 3.42-3.47 (t,2H, J=7 Hz), 3.58-3.77 (m, 17H), 3.81-4.87 (m, 1H), 4.17 (s, 2H),4.19-4.22 (m, 3H). MS (ESI-MS) calcd for C23H44Br2O7 592.40, found 615.2[M+Na]+.

A solution of dibromine (compound 3) (100 mg, 0.17 mmol) and K2CO3 (117mg, 0.85 mmol) in acetone (10 mL) was added thioacetic acid (129 mg, 1.7mmol). The reaction mixture was stirred at room temperature overnight,(compound 4) 1H NMR (400 MHz, CDCl,) 1.09-1.35 (m, 13H), 1.48-1.68 (m,4H), 1.93-2.01 (m, 2H), 2.32 (s, 6H), 3.08-3.28 (m, 1H), 3.39-3.50 (m,3H), 3.55-3.78 (m, 17H), 4.15 (s, 2H), 4.18-4.24 (m, 2H). MS (ESI-MS)calcd for C27H50O9S2 582.81, found 621.3 [M+K]+.

The solution of diactyl-OEt (compound 4) in ethyl alcohol was then addedconcentrated hydrochloric acid and stirred overnight to provide freethiol compound, (compound 5) 1H NMR (400 MHz, CDCl,) 1.18-1.38 (m, 10H),1.49-1.61 (m, 8H), 2.72-2.98 (m, 3H), 3.36-3.42 (t, 2H, J=7 Hz),3.55-3.77 (m, 16H), 4.17 (s, 2H). MS (ESI-MS) calcd for C21H42O7S2470.68, found 471.3 [M+H]+.

B. Determining the Ratio of AuNP to Surface Conjugated SipA Proteins

To determine the ratio of AuNP to corona SipA proteins, the inventorsexposed the SipA-AuNP (7.2 pmoles) sample to sodium cyanide, whichdecomposes the gold particle core. This afforded solution was thendialyzed for one day using a Slide-A-Lyzer MINI dialysis unit(MW=10,000), and thereafter concentrated to 45 μl. This sample and thesame volume Sip A only (45 μl, 540.7 μg/ml) sample are trypsin digestedand measured with an Agilent Q-TOF 6538 mass spectrometer coupled withan Agilent HPLC 1200, Peptide IPEPAAGPVPDGGK ([M+2H]2+, m/z 652,8505)from the SipA protein is identified through MS/MS spectral match, andchosen for as surrogate for protein quantification. The extracted ionchromatography (EIC) peaks for this peptide from the aforementioned twosamples are integrated and compared. (FIG. 9). The ratio proteinonly/protein from SipA-AuNP is 7.5 based on the integrated areas. Thusthe total amount of SipA from SipA-AuNP can be determined by thefollowing equation:(45 μl×540.7 μg/ml)/7.5=3.24 μg or3.24 μg/74,000 g/mol (MW of SipA)=43.8 pmolesThe ratio of AuNP and conjugated SipA was thus estimated to be 1:6 (7.2pmols vs. 43.8 pmols).

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Each and every publication and patent mentioned in the abovespecification is herein incorporated by reference in its entirety forall purposes. Various modifications and variations of the describedmethods and system of the invention will be apparent to those skilled inthe art without departing from the scope and spirit of the invention.Although the invention has been described in connection with specificembodiments, the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art and in fields related thereto are intended tobe within the scope of the following claims.

We claim:
 1. A method for reducing one or more symptoms of cancer in amammalian subject in need thereof, comprising administering to saidsubject (a) a composition comprising purified SipA operably conjugatedto a nanoparticle, and (b) one or more cytotoxins, said cancer isselected from the group consisting of colon cancer, breast cancer,intestinal cancer, and bladder cancer, and said administering reducesone or more symptoms of said cancer.
 2. The method of claim 1, whereinsaid cancer comprises cancer cells resistant to at least one cytotoxin.3. The method of claim 1, wherein said cancer comprises cancer cellsthat overexpress one or more of P-gp and p53 compared to a control cell.4. The method of claim 1, wherein said SipA is administered in an amountthat is effective in one or more of a) reducing the level of expressionof P-gp in cells of said cancer, b) reducing the level of un-cleavedP-gp in cells of said cancer, and c) increasing the level of expressionof PERP in cells of said cancer.
 5. The method of claim 1, wherein saidmethod further comprises determining the level of expression of P-gp incells of said cancer.
 6. The method of claim 1, said cytotoxin comprisesdoxorubicin.
 7. The method of claim 1, said cancer symptom comprisestumor volume.
 8. A method for reducing one or more symptoms of cancer ina mammalian subject in need thereof, comprising administering to saidsubject a composition comprising purified SipA operably conjugated to acytotoxin, said cancer is selected from the group consisting of coloncancer, breast cancer, intestinal cancer, and bladder cancer, and saidadministering reduces one or more symptoms of said cancer.
 9. The methodof claim 8, said cytotoxin comprises doxorubicin.
 10. The method ofclaim 8, said cancer symptom comprises tumor volume.
 11. A method forreducing one or more symptoms of cancer in a mammalian subject in needthereof, wherein said cancer is associated with cells that overexpressone or more of P-gp and p53, said method comprising administering tosaid subject (a) composition comprising purified SipA, conjugated to ananoparticle, and (b) one or more cytotoxin, said cancer is selectedfrom the group consisting of colon cancer, breast cancer, intestinalcancer, and bladder cancer, said SipA is in an amount that is effectivein one or more of a) reducing the level of expression of P-gp in saidcells, b) reducing the level of un-cleaved P-gp in said cells, and c)increasing the level of expression of PERP in said cells, and saidadministering reduces one or more symptoms of said cancer.
 12. Themethod of claim 11, said cytotoxin comprises doxorubicin.
 13. The methodof claim 11, said cancer symptom comprises tumor volume.
 14. A methodfor reducing one or more symptoms of cancer in a mammalian subject inneed thereof, wherein said cancer is associated with cells thatoverexpress one or more of P-gp and p53, said method comprisingadministering to said subject (a) a composition comprising purified SipAfused to human influenza hemagglutinin (HA), and (b) one or morecytotoxin, said cancer is selected from the group consisting of coloncancer, breast cancer, intestinal cancer, and bladder cancer, and saidSipA is in an amount that is effective in one or more of a) reducing thelevel of expression of P-gp in said cells, b) reducing the level ofun-cleaved P-gp in said cells, and c) increasing the level of expressionof PERP in said cells, and said administering reduces one or moresymptoms of said cancer.
 15. The method of claim 14, wherein said cancercomprises cancer cells resistant to at least one cytotoxin.
 16. Themethod of claim 14, wherein said method further comprises determiningthe level of expression of P-gp in cells of said cancer.
 17. The methodof claim 14, said cytotoxin comprises doxorubicin.
 18. The method ofclaim 14, said cancer symptom comprises tumor volume.